Methods for promoting wound healing and hair growth

ABSTRACT

The present invention generally relates to uses of glial cell line-derived growth factor (GDNF) in cutaneous wound healing and hair growth. Methods of effecting hair growth and/or wound healing which feature administration of GDNF, or a biologically active fragment thereof, to subjects, e.g., human subject, are disclosed herein. The invention relates also to formulations and kits for achieving the indicated pharmaceutical advantages.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/787,870, filed Mar. 15, 2013. The entire contentof the above-referenced patent application is incorporated herein bythis reference.

BACKGROUND OF THE INVENTION

Adult organisms contain several types of cells with remarkableregenerative potential when provided with appropriate chemical orphysical stimuli. Wound healing or wound repair is an example of asystem where multiple biological pathways are activated during theregeneration of the entire tissue. Skin, the largest organ of the bodyself-renews throughout adult life. Hair follicles, described as the“bone marrow of the skin”, are a source of numerous growth factors,cytokines and hormones that helps in remodeling the cutaneousenvironment (Schmidt-Ullrich and Paus (2005), BioEssays 27, 247-261).The role of several growth factors have been reported in the initiationof hair follicle development at embryonic stage but not much is knownabout their development in adult animals. The role of growth factors inskin biology, in particular, in wound repair or wound healing, has alsobeen reported. However, the exact role of certain growth factors andcytokines in complex processes such as wound repair or wound healing andhair growth remains to be elucidated. Such understanding would greatlyfacilitate the development of such growth factors and cytokines aspharmaceutical and/or therapeutic agents useful in these complexprocesses.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery thatthe cytokine, glial cell line-derived growth factor (GDNF) plays uniqueroles in the complex processes of wound repair and hair growth.Accordingly, the present invention relates to various pharmaceuticaland/or therapeutic methods that feature, in common, administration ordelivery of glial cell line-derived growth factor (GDNF) as a biologicactive agent. The invention is based, at least in part, on the discoveryof several important biological activities of GDNF. In particular, thepresent inventors have discovered significant regulatory roles for GDNFin biological processes including wound healing and hair growth.Accordingly, in one aspect, the invention relates to methods ofpromoting wound healing, in particular cutaneous wound healing, whereinsaid methods feature administration of GDNF, or a biologically activefragment thereof, to a wound site of a subject, e.g., a human subject,in a dose and/or for a time period sufficient to promote wound healing.In particular, the GDNF, or biologically active fragment thereof, isadministered in a dose and/or for a time sufficient to promote fillingand re-epithelialization of a wound site. In a related aspect, the GDNF,or biologically active fragment thereof, is administered in a doseand/or for a time sufficient to promote reestablishment of a skinbarrier at the wound site. In another aspect, the invention relates tomethods of promoting hair growth on a subject, wherein said methodsfeature administration of GDNF, or a biologically active fragmentthereof, at site of desired hair growth, in a dose and/or for a timesufficient to promote hair growth on the subject. In another aspect, theinvention features pharmaceutical formulations that include atherapeutically effective dose of isolated GDNF, or a biologicallyactive fragment thereof. In yet another aspect, the invention featureskits that include said pharmaceutical formulations. In yet anotheraspect, the invention features the use of a therapeutically effectivedose of GDNF, or a biologically active fragment thereof, for promotingwound healing at a wound site in a subject. In yet another aspect, theinvention features the use of a pharmaceutically effective dose of GDNF,or a biologically active fragment thereof, for promoting hair growth ata desired site in a subject.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Mice overexpressing Gdnf under the Catihespin L gene promoterhave altered hair follicle development. FVB transgenic miceTg(Ctsl-Gdnf) have ruffled fur by 3 wk of age (A). Skin sections stainedwith H&E from adult transgenic FVB mouse with more number of hairfollicles adjacent to normal hair development (asterisk) compared toskin section from control littermate (B). The transgene overexpressingGDNF in B6C3H background mice also show multiple layers of hairfollicles (C). Scale=100 μm.

FIG. 2. Subcutaneous injection of recombinant GDNF in adult mice resultsin an increase in hair follicles. Images of skin sections from B6 wildtype mice injected with 100 μl PBS or 10 μg GDNF on alternate days fortwo weeks. Scale=100 μm.

FIG. 3. Multiple injections of low dose are more effective than oneinjection of high concentration of GDNF. H&E image of skin from mouseinjected with 100 ul of 0.5 mg/ml (A) or 5 injections of 100 ul of 0.1mg/ml of GDNF on alternate days. Both images are at same magnification.Scale=100 um.

FIG. 4. New hair follicle development is specific to GDNF. Adult wildtype B6 mice injected with 50 μg FGF9 or GDNF and analyzed after 6 days.There is an increased number of new hair follicles in sections from miceinjected with GDNF. Scale=100 um.

FIG. 5. Increased number of hair follicles at a higher dose GDNF. Newhair follicles develop as early as day 9 in mice injected with 10 μg ofGDNF compared to mice injected with PBS (A). By day 9 there are multiplelayers of hair follicles in mice injected with 50 μg GDNF compared onesinjected with 10 μg (B). Ki67 labeled cells in hair follicles of skinsections injected with GDNF (C).

FIG. 6. Time course of hair follicle stimulation following GDNFtreatment. Hematoxylin and eosin stained section of skin, injected(s.c.) with PBS or 50 μg of GDNF protein and analyzed after 9, 13, and20 days. There are more numbers of hair follicles at the GDNF injectedsite compared to PBS. Scale=100 um

FIG. 7. GDNF seems to be activating Notch1 signaling pathway. RT-PCRanalysis on RNA extracted after 6 days from skin samples injected with50 μg of GDNF or PBS in B6 wild type mice. Three fold increase in Notch1but not c-Myc transcript was detected in GDNF injected sample.Transcript of Ret, the tyrosine kinase receptor, protein known to be inGDNF-GFRα1 signaling complex, is 4 fold higher in GDNF injected skin.

FIG. 8A-8B. GDNF accelerates wound-healing process in B6 wild type mice.Equal size 3 mm of wounds were made by biopsy punch needle on the dorsalside of adult mice. Wounds were photographed on day 0 and after oneweek. Only one wound site was injected with GDNF (arrow). H&E stainedsections of wound sites injected with PBS or one injection of 0.1 ml of0.5 mg/ml of GDNF. By one week all layers of skin, epidermis, dermis andhair follicles are present at the wound site injected with GDNF comparedto PBS injected sites (A). In the GDNF injected site, a complete layerof epithelium is seen throughout the wound site (arrow) and new bloodvessels (arrowheads) after 96 hr were as red blood cells are seen at PBSinjected site (asterisk) (B).

FIG. 9. GDNF accelerate the wound healing process in diabeticBKS.Cg-Dock7^(m)+/+Lepr^(db)/J mice. In the diabetic mice,re-epithelialization (arrow) and neovascularization and blood vessels(arrowhead) formation is observed as early as 96 hr. However, completewound healing occurs after two weeks in the mice injected with 125 μg ofGDNF. Scale=100 um

FIG. 10. GDNF induces blood vessel growth in FVB mice. The figure showsan increased blood vessel growth and branching after injection of 50 μgGDNF (B) in comparison to the PBS control (A).

DETAILED DESCRIPTION OF THE INVENTION

Glial Cell-Derived Neurotrophic Factor (GDNF) is a growth factor with40% sequence homology to the TGFβ superfamily. First identified assurvival factor for dopaminergic neurons of mid-brain and shown to beimportant for the development and maintenance of neural and othertissues (Lin et al 1993, Science 260, 1130-2). GDNF is part of complexthat include the high affinity receptor tyrosine kinase, c-RET andglycosyphatidylinositol (GPI-) linked co-receptor, GFRα1 that activatesspecific signaling pathway leading to cell proliferation, survival, andother differentiation effects.

The present invention is based, at least in part, on the discovery ofcertain key regulatory roles for GDNF in complex processes includingwound healing and hair growth. In initial experiments, the effect ofGDNF on hair follicle growth was studied. For these studies, purifiedactive recombinant protein expressed in baculovirus (as mammalianproteins expressed in baculovirus are glycosylated) was injectedsubcutaneously in mice and assayed for hair follicle growth. Preliminaryresults showed increase in number of hair follicles in mice injectedwith active form of GDNF compared to mice injected with PBS alone. Thisfinding was very surprising, especially as the effect could be seenafter a single injection.

The cutaneous wound healing process involves four steps, first theinflammatory phase followed by proliferative phase, the remodeling stepand finally the epithelialization, when new skin is formed. During theproliferative phase extracellular matrix (ECM) components aresynthesized and new blood vessels are formed on the matrix. Genesinvolved in the inflammatory response, angiogenesis and response towounding were differentially expressed in tissues over-expressing Gdnf.Moreover, when GDNF was injected subcutaneously into mice, smoother skinwas discovered. It was therefore predicted that GDNF influences the skinremodeling during wound healing. These findings, in addition to thedetailed studies presented in the Examples Section, infra., lead theinventors to propose GDNF as novel factor for hair growth, woundhealing, and treatment of scars, wrinkles (anti-aging), in particular,when the GDNF is applied topically to skin.

Prior to describing the invention, it may be helpful to an understandingthereof to set forth definitions of certain terms to be usedhereinafter.

I. Definitions

The terms “homolog,” “paralog,” and “ortholog,” have their artrecognized meanings. Typically, a homolog of a given gene product is oneof functional similarity as well as sequence similarity. If the homologis derived from a different organism, the homolog may be referred to asthe ortholog. If several homologs exist in a given organism, the homologmay be referred to as a paralog. Typically, the sequencesimilarity/identity between homologs is at least about 40%, 50%, 60%,70%, 80%, 90%, or more (or a percentage falling within any interval orrange of the foregoing). Methods for determining suchsimilarity/identity are described, infra. Domains (e.g., TGFβ-likedomains) conserved between homologs can have a sequencesimilarity/identity of at least about 70%, 80%, 90%, or more. It isunderstood that when comparing gene product sequence between diverseorganisms, for example, flies and humans, sequence similarity betweengiven homologs (e.g., orthologs) across the entire protein sequence maybe low. However, if functional complementarity exists, and in addition,if conserved domains exist, e.g., TGFβ-like domains, then the geneproducts being compared can be considered homologs and thus selected ascompositions for use in the methods of the invention, as describedherein.

The term “bioactive fragment” includes any portion (e.g., a segment ofcontiguous amino acids) of a polypeptide, e.g., a GDNF polypeptide orortholog thereof, sufficient to exhibit or exert at least activity ofthe polypeptide, e.g., at least one GDNF-associated activity including,for example, a growth promoting activity.

The phrase “encodes a gene product” includes the generation of a RNAmolecule from a DNA molecule (i.e., a complementary RNA moleculegenerated from the DNA molecule by the process of transcription) and/orthe generation of a polypeptide or protein molecule from an RNA (i.e.,by the processes of transcription and translation).

The term “expression” of a gene or nucleic acid encompasses not onlycellular gene expression, but also the transcription and translation ofnucleic acid(s) in cloning systems and in any other context.

The term “subject”, as used herein, includes living organisms. Examplesof subjects include humans, monkeys, cows, sheep, goats, horses, camels,dogs, cats, mice, rats, and transgenic species thereof. Administrationof the compositions of the present invention to a subject to be treatedcan be carried out using known procedures, at dosages and for periods oftime effective to modulate hair growth and/or wound healing in thesubject as further described herein.

As used herein, the term “isolated” molecule (e.g., isolated proteinmolecule or isolated peptide) refers to molecules which aresubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized.

The term “treatment”, as used herein, is defined as the application oradministration of a therapeutic agent to a subject, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a subject, who has a disease or disorder, a symptom of a disease ordisorder, or a predisposition toward a disease or disorder, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect the disease or disorder, the symptoms of the diseaseor disorder, or the predisposition toward a disease or disorder. Atherapeutic agent includes, but is not limited to, GDNF peptides,proteins, protein fragments, peptidomimetics, and the like.

The term “effective amount”, as used herein, is defined as that amountnecessary or sufficient to treat or prevent a disorder. The “effectiveamount” can vary depending on such factors as the size and weight of thesubject, the type of illness, or the particular agent beingadministered. One of ordinary skill in the art would be able to studythe aforementioned factors and make the determination regarding theeffective amount of the agent without undue experimentation.

The term “pharmaceutical composition” as used herein, refers to an agentformulated with one or more compatible solid or liquid filler diluentsor encapsulating substances, which are suitable for administration to ahuman or lower animal.

The term “administering” refers to an administration that is oral,topical, intravenous, subcutaneous, transcutaneous, transdermal,intramuscular, intra-joint, parenteral, intra-arteriole, intradermal,intraventricular, intracranial, intraperitoneal, intralesional,intranasal, rectal, vaginal, by inhalation or via an implantedreservoir. The term “parenteral” includes subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional, and intracranial injections orinfusion techniques. Typically, the treatment compositions of thepresent invention are administered topically or by subcutaneousinjection.

A “suitable control” or “appropriate control” refers to any control orstandard familiar to one of ordinary skill in the art useful forcomparison purposes.

The term “cell” refers to any cell of a biological organism. Preferredcells are eukaryotic cells, including but not limited to, animal cells(e.g., mammalian cells, e.g., human or murine cells), nematode cells,plant cells, and yeast. The term includes cell lines, e.g., transformedmammalian cell lines as well as embryonic cells, e.g., embryonic stemcells. Eukaryotic cells responsive to GDNF, or eukaryotic cells involvedin hair growth and/or wound repair are preferred cells of the invention.Also contemplated for use in the invention are prokaryotic cells, foruse, for example, in methods of manufacturing proteins, e.g., GDNF.

The term “tissue” refers to a collection of cells, usually of differentcell types, organized in a manner that imparts complex biologicalactivity.

The term “cell extract” refers to a lysate or acellular preparation of acell as defined above and can be a crude extract or partially purifiedas well as comprise additional agents such as recombinant polypeptides,nucleic acids, and/or buffers or stabilizers.

The term “organism” refers to multicellular organisms such as, e.g., C.elegans, Drosophila, mouse, and human.

The terms used herein are not intended to be limiting of the invention.

II. Glial Cell Line-Derived Neurotrophic Factor (GDNF)

Glial cell line-derived neurotrophic factor (or glial cell-derivedneurotrophic factor) (GDNF), also known as ATF1, ATF2, HSCR3, andHFB1-GDNF is a distant member of the TGF-β superfamily.Glial-cell-line-derived neurotrophic factor (GDNF) was originallyidentified as a survival factor for midbrain dopaminergic neurons, andwas able to prevent apoptosis of motor neurons induced by axotomy. GDNFand related ligands, neurturin (NRTN), artemin (ARTN) and persephin(PSPN), maintain several neuronal populations in the central nervoussystems, including midbrain dopamine neurons and motorneurons. Inaddition, GDNF, NRTN and ARTN support the survival and regulate thedifferentiation of many peripheral neurons, including sympathetic,parasympathetic, sensory and enteric neurons. GDNF has further criticalroles outside the nervous system in the regulation of kidneymorphogenesis and spermatogenesis.

GDNF family ligands bind to specific GDNF family receptor alpha(GFRalpha) proteins, all of which form receptor complexes and signalthrough the RET receptor tyrosine kinase (the product of the c-ret(rearranged during transfection) protooncogene). The biological activityof GDNF is mediated by its corresponding high affinity receptor, GDNFfamily receptor a-1 (GFRα-1) which functions as part of a receptorcomplex with the intracellular-signaling component, RET. The mature formof the protein is considered a ligand for RET. GDNF also showslower-affinity interactions with GFRα-2. The biology of GDNF signalingis much more complex than originally assumed. The neurotrophic effect ofGDNF, except in motorneurons, requires the presence of TGF-β, whichactivates the transport of GFRα1 to the cell membrane. GDNF can alsosignal RET independently through GFR1α. Upon ligand binding, GDNF incomplex with GFRα1 may interact with heparan sulphate glycosaminoglycansto activate the Met receptor tyrosine kinase through cytoplasmicSrc-family kinases. GDNF family ligands also signal through the neuralcell adhesion molecule NCAM. In cells lacking RET, GDNF binds with highaffinity to the NCAM and GFRα1 complex, which activates Fyn and FAK.

This GDNF gene encodes a highly conserved neurotrophic factor. Theencoded protein is processed to a mature secreted form that exists as ahomodimer. Multiple transcript variants encoding different isoforms havebeen found for the human GDNF gene. Transcript variant (1) differs inthe 5′ UTR, representing use of an alternate promoter, and a downstreamstart codon, compared to variant 3. The resulting isoform (1) has ashorter N-terminus, compared to isoform 3. Transcript variant (2) alsodiffers in the 5′ UTR, and represents use of an alternate promoter, usesa downstream start codon, and uses an alternate in-frame splice site inthe coding region, compared to variant 3. The resulting isoform (2) hasa shorter N-terminus and lacks an internal segment, compared to isoform3. Transcript variant (3) represents the longest transcript and encodesthe longest isoform (3). Transcript Variant: This variant (4) uses analternate in-frame splice site in the coding region, compared to variant3. The resulting isoform (4) lacks an internal segment, compared toisoform 3.

The nucleic acids encoding the human GDNF isoforms are as follows:

<gi|299473777|ref|NM_000514.3| Homo sapiens glialcell derived neurotrophic factor (GDNF), trans- cript variant 1, mRNA(SEQ ID NO: 1) CCGCCTCCAGCGCGCCCTTGCTGCCCCGCGCGACCCCAGGATTGCGAACTCTTGCCCCTGACCTGTTGGGCGGGGCTCCGCGCTCCAGCCATCAGCCCGGATGGGTCTCCTGGCTGGGACTTGGGGCACCTGGAGTTAATGTCCAACCTAGGGTCTGCGGAGACCCGATCCGAGGTGCCGCCGCCGGACGGGACTTTAAGATGAAGTTATGGGATGTCGTGGCTGTCTGCCTGGTGCTGCTCCACACCGCGTCCGCCTTCCCGCTGCCCGCCGGTAAGAGGCCTCCCGAGGCGCCCGCCGAAGACCGCTCCCTCGGCCGCCGCCGCGCGCCCTTCGCGCTGAGCAGTGACTCAAATATGCCAGAGGATTATCCTGATCAGTTCGATGATGTCATGGATTTTATTCAAGCCACCATTAAAAGACTGAAAAGGTCACCAGATAAACAAATGGCAGTGCTTCCTAGAAGAGAGCGGAATCGGCAGGCTGCAGCTGCCAACCCAGAGAATTCCAGAGGAALAGGTCGGAGAGGCCAGAGGGGCAAAAACCGGGGTTGTGTCTTAACTGCAATACATTTAAATGTCACTGACTTGGGTCTGGGCTATGAAACCAAGGAGGAACTGATTTTTAGGTACTGCAGCGGCTCTTGCGATGCAGCTGAGACAACGTACGACAAAATATTGAAAAACTTATCCAGAAATAGAAGGCTGGTGAGTGACAAAGTAGGGCAGGCATGTTGCAGACCCATCGCCTTTGATGATGACCTGTCGTTTTTAGATGATAACCTGGTTTACCATATTCTAAGAAAGCATTCCGCTAAAAGGTGTGGATGTATCTGACTCCGGCTCCAGAGACTGCTGTGTATTGCATTCCTGCTACAGTGCAAAGAAAGGGACCAAGGTTCCCAGGAAATGTTTGCCCAGAATGGAAGATGAGGACCAAGGAGGCGGAGGAGGAGGAAGAAGAAGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAAGGCAGCCATCATGGGAGCCTGGTAGAGGGAGATCCAGCTACAGACAACTGGACAGGAGAGAGAGAAAACAGCCCTCTGGATTCTCCAGGATGGCAGCCGATGTCACTAGAAGCTCAGGGCTGATGTTCCTGGTTGGCTATTGCCACCATTTCAGCTGATACAGTCCACCATCACTGATTACCGGCGCGGTTGCGGTGGATGCACTTGAACCAAACCAGTGTATCTCCTGTGATTTGTTTTCATGTGTCCGAAGACACCAGGGAAACAGAGATCCTGGTGTTGTTCCTTGTTATTACGTTTTACTGCTGAAAGTAAGAGGTTTATTTTTCTGTCACTCAGTGGAGACATACCCTGGAAAGGAGAGGGGAAAAAAAAAGCAAAGATACAAGAGATAATCACCTTTGCATTTGAAAGTTGAGGCCCGAGGTTTACTACAACCAGCATTTTTGCCAACGGTTGGTGATTGATTTCCATCACGGTGTGTGGGGTGGGAAGAAGTTGGCTAGGAACCAAAAAGGCTGTGCTCATGATTAAACACAAACCTGAAGGTATTTCCTTTATGTCCTTGGAAACAGGAAACGAGTTGTGGTTTTCGCCAGCATTCTTGTAGGAGAGAATCGGGGAAGGCCCCGAACTGCCCCCGGGCAGGGAGAGCCCCTCAGGCCTGTTGGTTTACAGAGAGACAGATGTTACATAACCAGCTCCGTTGATGCGTGGTCACCAGTGACCAGAGAAGCTACTCGATGCAATGCATCTGTTTCAGATACAGAAATATAGAGAAGATATTTATTGAAATTTAAGTTATTGTTATTTATTACCGTTCACTAATGAATTTCTCTTTTTTCCCTTATTTATTAAAGTTTCTTTTCAAAGGTGCCAAAGTATATGTGCTCGCAAAATGCAAAGAAAGGTGACAAAAGGAAATTTGAATTGGGAACAAGGGTCCATGCTTTTCAAAGTATTAAAAAGTTTTTTGCCAGGCAAAAATCACTTACTTTACCTTTTTAAGAAAATTTGTCATTAATTTTCCCCAGATTTCAGCATTTTTCCCAATTTTTATTTGTGGAGCATCTCAGGCAAGCCCCCTTTCCTGGAGCAGCGTGCAGAGACCACTGGCACTTGACTTTATTTCTTCCTTGCTCCATTGCTGAACAGAAATGTCGTGGGCTCCACTTCCTGTTGTCTTTAAGCTCTTAGTCCCCTCCACGTATACCTATCTGTACTATGCATAACCATATGTAGAAAAGGTTCAGTTCCTTTTAGTAGGTAGTCCTGGATTTAATGCTGACCTAAAAGTAATGTCGACAATGCTGTCAGGTAGCTGCCGTTCTACCGACTCCCTCCATCCCTGCCCACCCACTGCCCTCCCGAGAATATGCTGGCTGCCCAGTGCAGCCCGGGAGACACAGGGGCCTTCCAGAGGTAGGGTCTACCAGGTCCTGTACAACCCCTGGGCTGTCACCGGGGGTCAACAGCTGCTGCTCCTATATACCCAAACACCTGACAGCTCCCTGGGGAGCAGATGGCTGAGAAGGGTGCTGAGGAAGCCATATTGGGACCAGCCACAGCCACACACATGGAGCCTCATACTTAGGAGCGTGCTGCCTTTAAATGAAGGTGGTCGGGGCCAGTGCAGCGGCTCACACCCATAATCCCAACACTTTGGAAAGCCAAGGTGGGAGGATCTCTTGAACCCAGGAGTTTGAGACCAGCTTGGGCAACATAGGGAGACCCTGTCTCTACAGAAACTTTAAAAATTAGGCAGGCATGATGGTGCACACCTGTGGTCCCAGCTACTCAAGAGGCTGAAGGAGGATCACTTGAGTCCAGAAGGTCGAGGCTGCAGTGAGCTGTGATCATGCCACTGCACTCCAGCCTAAGTGACAGTGCGGTACCCTGTCTCAAAAAAAAAAAAAAAAAAAAAAAGAGGTTGGAGCAGGAGGAAGCATAGGGGCGGGAACAGCCACCTCCTCCATGCCCTAGATTGTGAATTTATCGGGCAGCCAACACATGTATGACACACTAGGCCCTGTATTACAGCTTGTTACGCATTTCATAAAAGGGATTTTCATTAAGGAGATAATCTATTACTACCTACCTTAGTGGCTACTAGTATAAAACTATGACAGATTTAGCAATTAAATGAAATACTGGCCTCCATCAAATAATCATAGTAACAAGAAGCAGCAGTTACCAGACATCTGATCCCCTTCCCCCAAAATACCCAAATTCTTCATGGTTCTGCCCTTCTCTGTCCTTTCTGCTCCCCTTGCTCGCCTGGGAAATGGAGGAAAGGCCTTCCCTCTCACACTGTCTTGGGATCTTGCTGAGAATTCAGACTGCTCGAAACAGTGACAAACCCCAGCCATCCAGTCATTCGTGGAGCACAATTTGGATGTGGCCCCAGGGGCATCTGTCCCATTCAGAGAACCTTGGCAGTGCGATGGCCACTGTTCCCAGGCTTCAACCTCAGTGACCCCCCCCAACAACTCCCCATGGAGAGTCCCTGCCCAAAAAAGCTGTAGGATCCAAGGGGTGTCAATAGCTCGTTCCCGGCATCACCTACACACCACAAGCAGGTTTTAATGGAAGCAAGTTGCTCCACCAAATCCACAAAAGGGTAAAGTTTGTGATTTTTCTTTATCATTGCGATCACCATCTGATACCGTAAGGAGTGCACTTGTTTGGAAGTTCTGACTTCTCTGATCTGTCTTGGTCGTTTGTGTTATAAAACCAAAGTTCTCTACAGACTTTATTTTTGTACAATATCATTTTGTAACTTTTTACAAATAAAAACTCA TTTCTATTGC<gi|299473776|ref|NM_199231.2| Homo sapiens glialcell derived neurotrophic factor (GDNF), trans- cript variant 2, mRNA(SEQ ID NO: 2) CATACAGGCCAAAAGTCTCCAAGTCCCTGCTAACTTCTTGCTCTCGCAACAGAATACCTATTTAGGTGGGAAGAATGAGGTGTGGGCGGCAGGCTGGGTGCCGCCGCCGGACGGGACTTTAAGATGAAGTTATGGGATGTCGTGGCTGTCTGCCTGGTGCTGCTCCACACCGCGTCCGCCTTCCCGCTGCCCGCCGCAAATATGCCAGAGGATTATCCTGATCAGTTCGATGATGTCATGGATTTTATTCAAGCCACCATTAAAAGACTGAAPAGGTCACCAGATAAACAAATGGCAGTGCTTCCTAGAAGAGAGCGGAATCGGCAGGCTGCAGCTGCCAACCCAGAGAATTCCAGAGGAAAAGGTCGGAGAGGCCAGAGGGGCAAAAACCGGGGTTGTGTCTTAACTGCAATACATTTAAATGTCACTGACTTGGGTCTGGGCTATGAAACCAAGGAGGAACTGATTTTTAGGTACTGCAGCGGCTCTTGCGATGCAGCTGAGACAACGTACGACAAAATATTGAAAAACTTATCCAGAAATAGAAGGCTGGTGAGTGACAAAGTAGGGCAGGCATGTTGCAGACCCATCGCCTTTGATGATGACCTGTCGTTTTTAGATGATAACCTGGTTTACCATATTCTAAGAAAGCATTCCGCTAAAAGGTGTGGATGTATCTGACTCCGGCTCCAGAGACTGCTGTGTATTGCATTCCTGCTACAGTGCAAAGAAAGGGACCAAGGTTCCCAGGAAATGTTTGCCCAGAATGGAAGATGAGGACCAAGGAGGCGGAGGAGGAGGAAGAAGAAGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAAGGCAGCCATCATGGGAGCCTGGTAGAGGGAGATCCAGCTACAGACAACTGGACAGGAGAGAGAGAAAACAGCCCTCTGGATTCTCCAGGATGGCAGCCGATGTCACTAGAAGCTCAGGGCTGATGTTCCTGGTTGGCTATTGCCACCATTTCAGCTGATACAGTCCACCATCACTGATTACCGGCGCGGTTGCGGTGGATGCACTTGAACCAAACCAGTGTATCTCCTGTGATTTGTTTTCATGTGTCCGAAGACACCAGGGAAACAGAGATCCTGGTGTTGTTCCTTGTTATTACGTTTTACTGCTGAAAGTAAGAGGTTTATTTTTCTGTCACTCAGTGGAGACATACCCTGGAAAGGAGAGGGGAAAAAAAAAGCAAAGATACAAGAGATAATCACCTTTGCATTTGAAAGTTGAGGCCCGAGGTTTACTACAACCAGCATTTTTGCCAACGGTTGGTGATTGATTTCCATCACGGTGTGTGGGGTGGGAAGAAGTTGGCTAGGAACCAAAAAGGCTGTGCTCATGATTAAACACAAACCTGAAGGTATTTCCTTTATGTCCTTGGAAACAGGAAACGAGTTGTGGTTTTCGCCAGCATTCTTGTAGGAGAGAATCGGGGAAGGCCCCGAACTGCCCCCGGGCAGGGAGAGCCCCTCAGGCCTGTTGGTTTACAGAGAGACAGATGTTACATAACCAGCTCCGTTGATGCGTGGTCACCAGTGACCAGAGAAGCTACTCGATGCAATGCATCTGTTTCAGATACAGAAATATAGAGAAGATATTTATTGAAATTTAAGTTATTGTTATTTATTACCGTTCACTAATGAATTTCTCTTTTTTCCCTTATTTATTAAAGTTTCTTTTCAAAGGTGCCAAAGTATATGTGCTCGCAAAATGCAAAGAAAGGTGACAAAAGGAAATTTGAATTGGGAACAAGGGTCCATGCTTTTCAAAGTATTAAAAAGTTTTTTGCCAGGCAAAAATCACTTACTTTACCTTTTTAAGAAAATTTGTCATTAATTTTCCCCAGATTTCAGCATTTTTCCCAATTTTTATTTGTGGAGCATCTCAGGCAAGCCCCCTTTCCTGGAGCAGCGTGCAGAGACCACTGGCACTTGACTTTATTTCTTCCTTGCTCCATTGCTGAACAGAAATGTCGTGGGCTCCACTTCCTGTTGTCTTTAAGCTCTTAGTCCCCTCCACGTATACCTATCTGTACTATGCATAACCATATGTAGAALAGGTTCAGTTCCTTTTAGTAGGTAGTCCTGGATTTAATGCTGACCTAAAAGTAATGTCGACAATGCTGTCAGGTAGCTGCCGTTCTACCGACTCCCTCCATCCCTGCCCACCCACTGCCCTCCCGAGAATATGCTGGCTGCCCAGTGCAGCCCGGGAGACACAGGGGCCTTCCAGAGGTAGGGTCTACCAGGTCCTGTACAACCCCTGGGCTGTCACCGGGGGTCAACAGCTGCTGCTCCTATATACCCAAACACCTGACAGCTCCCTGGGGAGCAGATGGCTGAGAAGGGTGCTGAGGAAGCCATATTGGGACCAGCCACAGCCACACACATGGAGCCTCATACTTAGGAGCGTGCTGCCTTTAAATGAAGGTGGTCGGGGCCAGTGCAGCGGCTCACACCCATAATCCCAACACTTTGGAAAGCCAAGGTGGGAGGATCTCTTGAACCCAGGAGTTTGAGACCAGCTTGGGCAACATAGGGAGACCCTGTCTCTACAGAAACTTTAAAAATTAGGCAGGCATGATGGTGCACACCTGTGGTCCCAGCTACTCAAGAGGCTGAAGGAGGATCACTTGAGTCCAGAAGGTCGAGGCTGCAGTGAGCTGTGATCATGCCACTGCACTCCAGCCTAAGTGACAGTGCGGTACCCTGTCTCAAAAAAAAAAAAAAAAAAAAAAAGAGGTTGGAGCAGGAGGAAGCATAGGGGCGGGAACAGCCACCTCCTCCATGCCCTAGATTGTGAATTTATCGGGCAGCCAACACATGTATGACACACTAGGCCCTGTATTACAGCTTGTTACGCATTTCATAAAAGGGATTTTCATTAAGGAGATAATCTATTACTACCTACCTTAGTGGCTACTAGTATAAAACTATGACAGATTTAGCAATTAAATGAAATACTGGCCTCCATCAAATAATCATAGTAACAAGAAGCAGCAGTTACCAGACATCTGATCCCCTTCCCCCAAAATACCCAAATTCTTCATGGTTCTGCCCTTCTCTGTCCTTTCTGCTCCCCTTGCTCGCCTGGGAAATGGAGGAAAGGCCTTCCCTCTCACACTGTCTTGGGATCTTGCTGAGAATTCAGACTGCTCGAAACAGTGACAAACCCCAGCCATCCAGTCATTCGTGGAGCACAATTTGGATGTGGCCCCAGGGGCATCTGTCCCATTCAGAGAACCTTGGCAGTGCGATGGCCACTGTTCCCAGGCTTCAACCTCAGTGACCCCCCCCAACAACTCCCCATGGAGAGTCCCTGCCCAAAAAAGCTGTAGGATCCAAGGGGTGTCAATAGCTCGTTCCCGGCATCACCTACACACCACAAGCAGGTTTTAATGGAAGCAAGTTGCTCCACCAAATCCACAAAAGGGTAAAGTTTGTGATTTTTCTTTATCATTGCGATCACCATCTGATACCGTAAGGAGTGCACTTGTTTGGAAGTTCTGACTTCTCTGATCTGTCTTGGTCGTTTGTGTTATAAAACCAAAGTTCTCTACAGACTTTATTTTTGTACAATATCATTTTGTAACTTTTTACAAATAAAAACTCATTTCT ATTGC<gi|299473778|ref|NM_001190468.1| Homo sapiensglial cell derived neurotrophic factor (GDNF),transcript variant 3, mRNA (SEQ ID NO: 3)CCAAAGCGTCCGAGACTGGGTACAGTCGTCCAGGCGTGACGGGGGCGCGGGGAGCCAGTGACTCCTCTGGGAGGGGAAGGGATTAGGGCCAGAATCTCTCAAAGGTGCAAAAATCCAGTCAAGAGAGGGTTTTCGGGTATACCACGGAGGATTAAAACTTTCAAGACAAATGCAGTCTTTGCCTAACAGCAATGGTGCCGCCGCCGGACGGGACTTTAAGATGAAGTTATGGGATGTCGTGGCTGTCTGCCTGGTGCTGCTCCACACCGCGTCCGCCTTCCCGCTGCCCGCCGGTAAGAGGCCTCCCGAGGCGCCCGCCGAAGACCGCTCCCTCGGCCGCCGCCGCGCGCCCTTCGCGCTGAGCAGTGACTCAAATATGCCAGAGGATTATCCTGATCAGTTCGATGATGTCATGGATTTTATTCAAGCCACCATTAAAAGACTGAAAAGGTCACCAGATAAACAAATGGCAGTGCTTCCTAGAAGAGAGCGGAATCGGCAGGCTGCAGCTGCCAACCCAGAGAATTCCAGAGGAAAAGGTCGGAGAGGCCAGAGGGGCAAAAACCGGGGTTGTGTCTTAACTGCAATACATTTAAATGTCACTGACTTGGGTCTGGGCTATGAAACCAAGGAGGAACTGATTTTTAGGTACTGCAGCGGCTCTTGCGATGCAGCTGAGACAACGTACGACAALATATTGAAAAACTTATCCAGAAATAGAAGGCTGGTGAGTGACAAAGTAGGGCAGGCATGTTGCAGACCCATCGCCTTTGATGATGACCTGTCGTTTTTAGATGATAACCTGGTTTACCATATTCTAAGAAAGCATTCCGCTAAAAGGTGTGGATGTATCTGACTCCGGCTCCAGAGACTGCTGTGTATTGCATTCCTGCTACAGTGCAAAGAAAGGGACCAAGGTTCCCAGGAAATGTTTGCCCAGAATGGAAGATGAGGACCAAGGAGGCGGAGGAGGAGGAAGAAGAAGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAAGGCAGCCATCATGGGAGCCTGGTAGAGGGAGATCCAGCTACAGACAACTGGACAGGAGAGAGAGAAAACAGCCCTCTGGATTCTCCAGGATGGCAGCCGATGTCACTAGAAGCTCAGGGCTGATGTTCCTGGTTGGCTATTGCCACCATTTCAGCTGATACAGTCCACCATCACTGATTACCGGCGCGGTTGCGGTGGATGCACTTGAACCAAACCAGTGTATCTCCTGTGATTTGTTTTCATGTGTCCGAAGACACCAGGGAAACAGAGATCCTGGTGTTGTTCCTTGTTATTACGTTTTACTGCTGAAAGTAAGAGGTTTATTTTTCTGTCACTCAGTGGAGACATACCCTGGAAAGGAGAGGGGAAAALAAAAGCAAAGATACAAGAGATAATCACCTTTGCATTTGAAAGTTGAGGCCCGAGGTTTACTACAACCAGCATTTTTGCCAACGGTTGGTGATTGATTTCCATCACGGTGTGTGGGGTGGGAAGAAGTTGGCTAGGAACCAAAAAGGCTGTGCTCATGATTAAACACAAACCTGAAGGTATTTCCTTTATGTCCTTGGAAACAGGAAACGAGTTGTGGTTTTCGCCAGCATTCTTGTAGGAGAGAATCGGGGAAGGCCCCGAACTGCCCCCGGGCAGGGAGAGCCCCTCAGGCCTGTTGGTTTACAGAGAGACAGATGTTACATAACCAGCTCCGTTGATGCGTGGTCACCAGTGACCAGAGAAGCTACTCGATGCAATGCATCTGTTTCAGATACAGAAATATAGAGAAGATATTTATTGAAATTTAAGTTATTGTTATTTATTACCGTTCACTAATGAATTTCTCTTTTTTCCCTTATTTATTAAAGTTTCTTTTCAAAGGTGCCAAAGTATATGTGCTCGCAAAATGCAAAGAAAGGTGACAAAAGGAAATTTGAATTGGGAACAAGGGTCCATGCTTTTCAAAGTATTAAAAAGTTTTTTGCCAGGCAAAAATCACTTACTTTACCTTTTTAAGAAAATTTGTCATTAATTTTCCCCAGATTTCAGCATTTTTCCCAATTTTTATTTGTGGAGCATCTCAGGCAAGCCCCCTTTCCTGGAGCAGCGTGCAGAGACCACTGGCACTTGACTTTATTTCTTCCTTGCTCCATTGCTGAACAGAAATGTCGTGGGCTCCACTTCCTGTTGTCTTTAAGCTCTTAGTCCCCTCCACGTATACCTATCTGTACTATGCATAACCATATGTAGAAAAGGTTCAGTTCCTTTTAGTAGGTAGTCCTGGATTTAATGCTGACCTAAAAGTAATGTCGACAATGCTGTCAGGTAGCTGCCGTTCTACCGACTCCCTCCATCCCTGCCCACCCACTGCCCTCCCGAGAATATGCTGGCTGCCCAGTGCAGCCCGGGAGACACAGGGGCCTTCCAGAGGTAGGGTCTACCAGGTCCTGTACAACCCCTGGGCTGTCACCGGGGGTCAACAGCTGCTGCTCCTATATACCCAAACACCTGACAGCTCCCTGGGGAGCAGATGGCTGAGAAGGGTGCTGAGGAAGCCATATTGGGACCAGCCACAGCCACACACATGGAGCCTCATACTTAGGAGCGTGCTGCCTTTAAATGAAGGTGGTCGGGGCCAGTGCAGCGGCTCACACCCATAATCCCAACACTTTGGAAAGCCAAGGTGGGAGGATCTCTTGAACCCAGGAGTTTGAGACCAGCTTGGGCAACATAGGGAGACCCTGTCTCTACAGAAACTTTAAAAATTAGGCAGGCATGATGGTGCACACCTGTGGTCCCAGCTACTCAAGAGGCTGAAGGAGGATCACTTGAGTCCAGAAGGTCGAGGCTGCAGTGAGCTGTGATCATGCCACTGCACTCCAGCCTAAGTGACAGTGCGGTACCCTGTCTCAAAAAAAAAAAAAAAAAAAAAAAGAGGTTGGAGCAGGAGGAAGCATAGGGGCGGGAACAGCCACCTCCTCCATGCCCTAGATTGTGAATTTATCGGGCAGCCAACACATGTATGACACACTAGGCCCTGTATTACAGCTTGTTACGCATTTCATAAAAGGGATTTTCATTAAGGAGATAATCTATTACTACCTACCTTAGTGGCTACTAGTATAAAACTATGACAGATTTAGCAATTAAATGAAATACTGGCCTCCATCAAATAATCATAGTAACAAGAAGCAGCAGTTACCAGACATCTGATCCCCTTCCCCCAAAATACCCAAATTCTTCATGGTTCTGCCCTTCTCTGTCCTTTCTGCTCCCCTTGCTCGCCTGGGAAATGGAGGAAAGGCCTTCCCTCTCACACTGTCTTGGGATCTTGCTGAGAATTCAGACTGCTCGAAACAGTGACAAACCCCAGCCATCCAGTCATTCGTGGAGCACAATTTGGATGTGGCCCCAGGGGCATCTGTCCCATTCAGAGAACCTTGGCAGTGCGATGGCCACTGTTCCCAGGCTTCAACCTCAGTGACCCCCCCCAACAACTCCCCATGGAGAGTCCCTGCCCAAAAAAGCTGTAGGATCCAAGGGGTGTCAATAGCTCGTTCCCGGCATCACCTACACACCACAAGCAGGTTTTAATGGAAGCAAGTTGCTCCACCAAATCCACAAAAGGGTAAAGTTTGTGATTTTTCTTTATCATTGCGATCACCATCTGATACCGTAAGGAGTGCACTTGTTTGGAAGTTCTGACTTCTCTGATCTGTCTTGGTCGTTTGTGTTATAAAACCAAAGTTCTCTACAGACTTTATTTTTGTACAATATCATTTTGTAACTTTTTACAAATAAAAACTCATTTCTATTGC<gi|299473780|ref|NM_001190469.1| Homo sapiensglial cell derived neurotrophic factor (GDNF),transcript variant 4, mRNA (SEQ ID NO: 4)CCAAAGCGTCCGAGACTGGGTACAGTCGTCCAGGCGTGACGGGGGCGCGGGGAGCCAGTGACTCCTCTGGGAGGGGAAGGGATTAGGGCCAGAATCTCTCAAAGGTGCAAAAATCCAGTCAAGAGAGGGTTTTCGGGTATACCACGGAGGATTAAAACTTTCAAGACAAATGCAGTCTTTGCCTAACAGCAATGGTGCCGCCGCCGGACGGGACTTTAAGATGAAGTTATGGGATGTCGTGGCTGTCTGCCTGGTGCTGCTCCACACCGCGTCCGCCTTCCCGCTGCCCGCCGCAAATATGCCAGAGGATTATCCTGATCAGTTCGATGATGTCATGGATTTTATTCAAGCCACCATTAAAAGACTGAAAAGGTCACCAGATAAACAAATGGCAGTGCTTCCTAGAAGAGAGCGGAATCGGCAGGCTGCAGCTGCCAACCCAGAGAATTCCAGAGGAAAAGGTCGGAGAGGCCAGAGGGGCAAAAACCGGGGTTGTGTCTTAACTGCAATACATTTAAATGTCACTGACTTGGGTCTGGGCTATGAAACCAAGGAGGAACTGATTTTTAGGTACTGCAGCGGCTCTTGCGATGCAGCTGAGACAACGTACGACAAAATATTGAAALACTTATCCAGAAATAGAAGGCTGGTGAGTGACAAAGTAGGGCAGGCATGTTGCAGACCCATCGCCTTTGATGATGACCTGTCGTTTTTAGATGATAACCTGGTTTACCATATTCTAAGAAAGCATTCCGCTAAAAGGTGTGGATGTATCTGACTCCGGCTCCAGAGACTGCTGTGTATTGCATTCCTGCTACAGTGCAAAGAAAGGGACCAAGGTTCCCAGGAAATGTTTGCCCAGAATGGAAGATGAGGACCAAGGAGGCGGAGGAGGAGGAAGAAGAAGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAGGAAGGCAGCCATCATGGGAGCCTGGTAGAGGGAGATCCAGCTACAGACAACTGGACAGGAGAGAGAGAAAACAGCCCTCTGGATTCTCCAGGATGGCAGCCGATGTCACTAGAAGCTCAGGGCTGATGTTCCTGGTTGGCTATTGCCACCATTTCAGCTGATACAGTCCACCATCACTGATTACCGGCGCGGTTGCGGTGGATGCACTTGAACCAAACCAGTGTATCTCCTGTGATTTGTTTTCATGTGTCCGAAGACACCAGGGAAACAGAGATCCTGGTGTTGTTCCTTGTTATTACGTTTTACTGCTGAAAGTAAGAGGTTTATTTTTCTGTCACTCAGTGGAGACATACCCTGGAAAGGAGAGGGGAAAAAAAAAGCAAAGATACAAGAGATAATCACCTTTGCATTTGAAAGTTGAGGCCCGAGGTTTACTACAACCAGCATTTTTGCCAACGGTTGGTGATTGATTTCCATCACGGTGTGTGGGGTGGGAAGAAGTTGGCTAGGAACCAAAAAGGCTGTGCTCATGATTAAACACAAACCTGAAGGTATTTCCTTTATGTCCTTGGAAACAGGAAACGAGTTGTGGTTTTCGCCAGCATTCTTGTAGGAGAGAATCGGGGAAGGCCCCGAACTGCCCCCGGGCAGGGAGAGCCCCTCAGGCCTGTTGGTTTACAGAGAGACAGATGTTACATAACCAGCTCCGTTGATGCGTGGTCACCAGTGACCAGAGAAGCTACTCGATGCAATGCATCTGTTTCAGATACAGAAATATAGAGAAGATATTTATTGAAATTTAAGTTATTGTTATTTATTACCGTTCACTAATGAATTTCTCTTTTTTCCCTTATTTATTAAAGTTTCTTTTCAAAGGTGCCAAAGTATATGTGCTCGCAAAATGCAAAGAAAGGTGACAAAAGGAAATTTGAATTGGGAACAAGGGTCCATGCTTTTCAAAGTATTAAAAAGTTTTTTGCCAGGCAAAAATCACTTACTTTACCTTTTTAAGAAAATTTGTCATTAATTTTCCCCAGATTTCAGCATTTTTCCCAATTTTTATTTGTGGAGCATCTCAGGCAAGCCCCCTTTCCTGGAGCAGCGTGCAGAGACCACTGGCACTTGACTTTATTTCTTCCTTGCTCCATTGCTGAACAGAAATGTCGTGGGCTCCACTTCCTGTTGTCTTTAAGCTCTTAGTCCCCTCCACGTATACCTATCTGTACTATGCATAACCATATGTAGAAAAGGTTCAGTTCCTTTTAGTAGGTAGTCCTGGATTTAATGCTGACCTAAAAGTAATGTCGACAATGCTGTCAGGTAGCTGCCGTTCTACCGACTCCCTCCATCCCTGCCCACCCACTGCCCTCCCGAGAATATGCTGGCTGCCCAGTGCAGCCCGGGAGACACAGGGGCCTTCCAGAGGTAGGGTCTACCAGGTCCTGTACAACCCCTGGGCTGTCACCGGGGGTCAACAGCTGCTGCTCCTATATACCCAAACACCTGACAGCTCCCTGGGGAGCAGATGGCTGAGAAGGGTGCTGAGGAAGCCATATTGGGACCAGCCACAGCCACACACATGGAGCCTCATACTTAGGAGCGTGCTGCCTTTAAATGAAGGTGGTCGGGGCCAGTGCAGCGGCTCACACCCATAATCCCAACACTTTGGAAAGCCAAGGTGGGAGGATCTCTTGAACCCAGGAGTTTGAGACCAGCTTGGGCAACATAGGGAGACCCTGTCTCTACAGAAACTTTAAAAATTAGGCAGGCATGATGGTGCACACCTGTGGTCCCAGCTACTCAAGAGGCTGAAGGAGGATCACTTGAGTCCAGAAGGTCGAGGCTGCAGTGAGCTGTGATCATGCCACTGCACTCCAGCCTAAGTGACAGTGCGGTACCCTGTCTCAAAAAAAAAAAAAAAAAAAAAAAGAGGTTGGAGCAGGAGGAAGCATAGGGGCGGGAACAGCCACCTCCTCCATGCCCTAGATTGTGAATTTATCGGGCAGCCAACACATGTATGACACACTAGGCCCTGTATTACAGCTTGTTACGCATTTCATAAAAGGGATTTTCATTAAGGAGATAATCTATTACTACCTACCTTAGTGGCTACTAGTATAAAACTATGACAGATTTAGCAATTAAATGAAATACTGGCCTCCATCAAATAATCATAGTAACAAGAAGCAGCAGTTACCAGACATCTGATCCCCTTCCCCCAAAATACCCAAATTCTTCATGGTTCTGCCCTTCTCTGTCCTTTCTGCTCCCCTTGCTCGCCTGGGAAATGGAGGAAAGGCCTTCCCTCTCACACTGTCTTGGGATCTTGCTGAGAATTCAGACTGCTCGAAACAGTGACAAACCCCAGCCATCCAGTCATTCGTGGAGCACAATTTGGATGTGGCCCCAGGGGCATCTGTCCCATTCAGAGAACCTTGGCAGTGCGATGGCCACTGTTCCCAGGCTTCAACCTCAGTGACCCCCCCCAACAACTCCCCATGGAGAGTCCCTGCCCAAAAAAGCTGTAGGATCCAAGGGGTGTCAATAGCTCGTTCCCGGCATCACCTACACACCACAAGCAGGTTTTAATGGAAGCAAGTTGCTCCACCAAATCCACAAAAGGGTAAAGTTTGTGATTTTTCTTTATCATTGCGATCACCATCTGATACCGTAAGGAGTGCACTTGTTTGGAAGTTCTGACTTCTCTGATCTGTCTTGGTCGTTTGTGTTATAAAACCAAAGTTCTCTACAGACTTTATTTTTGTACAATATCATTTTGTAACTTTTTACAAATAAAAACTCATTTCTATT GC

A representative cDNA encoding isoform 1 is as follows:

(SEQ ID NO: 5) caaatatgccagaggattatcctgatcagttcgatgatgtcatggattttattcaagccaccattaaaagactgaaaaggtcaccagataaacaaatggcagtgcttcctagaagagagcggaatcggcaggctgcagctgccaacccagagaattccagaggaaaaggtcggagaggccagaggggcaaaaaccggggttgtgtcttaactgcaatacatttaaatgtcactgacttgggtctgggctatgaaaccaaggaggaactgatttttaggtactgcagcggctcttgcgatgcagctgagacaacgtacgacaaaatattgaaaaacttatccagaaatagaaggctggtgagtgacaaagtagggcaggcatgttgcagacccatcgcctttgatgatgacctgtcgtttttagatgataacctggtttaccatattctaagaaagcattccgctaaaaggtgtggatgtatctga

GenBank Accession No. L19063.1, gi:306761. See Science 1993, 260(5111):1130-2.

The amino acid sequences of the various human GDNF isoforms are asfollows:

<gi|40549411|ref|NP_954701.1| glial cell line-derived neurotrophic factor isoform 2 prepro- protein [Homo sapiens](SEQ ID NO: 6) MKLWDVVAVCLVLLHTASAFPLPAANMPEDYPDQFDDVMDFIQATIKRLKRSPDKQMAVLPRRERNRQAAAANPENSRGKGRRGQRGKNRGCVLTAIHLNVIDLGLGYETKEELIFRYCSGSCDAAETTYDKILKNLSRNRRLVSDKVGQACCRPIAFDDDLSELDDNLVYHILRKHSAKRCGCI<gi|4503975|ref|NP_000505.1| glial cell line-derived neurotrophic factor isoform 1 prepro- protein [Homo sapiens](SEQ ID NO: 7) MKLWDVVAVCLVLLHTASAFPLPAGKRPPEAPAEDRSLGRRRAPFALSSDSNMPEDYPDQFDDVMDFIQATIKRLKRSPDKQMAVLPRRERNRQAAAANPENSRGKGRRGQRGKNRGCVLTAIHLNVTDLGLGYETKEELIFRYCSGSCDAAETTYDKILKNLSRNRRLVSDKVGQACCRPIAFDDDLSFLDDNLVYHIL RKHSAKRCGCI<gi|299473779|ref|NP_001177397.1| glial cell line-derived neurotrophic factor isoform 3 prepro- protein [Homo sapiens](SEQ ID NO: 8) MQSLPNSNGAAAGRDFKMKLWDVVAVCLVLLHTASAFPLPAGKRPPEAPAEDRSLGRRRAPFALSSDSNMPEDYPDQFDDVMDFIQATIKRLKRSPDKQMAVLPRRERNRQAAAANPENSRGKGRRGQRGKNRGCVLTAIHLNVTDLGLGYETKEELIFRYCSGSCDAAETTYDKILKNLSRNRRLVSDKVGQACCRPIAFDDDLSFLDDNLVYHILRKHSAKRCGCI<gi|299473781|ref|NP_001177398.1| glial cell line-derived neurotrophic factor isoform 4 prepro- protein [Homo sapiens](SEQ ID NO: 9) MQSLPNSNGAAAGRDFKMKLWDVVAVCLVLLHTASAFPLPAANMPEDYPDQFDDVMDFIQATIKRLKRSPDKQMAVLPRRERNRQAAAANPENSRCKGRRGQRGKNRGCVLTAIHLNVTDLGLGYETKEELIFRYCSGSCDAAETTYDKILKNLSRNRRLVSDKVGQACCRPIAFDDDLSFLDDNLVYHILRKHSAKRCG CI

Like other growth factors GDNF has to be cleaved for secretion from thecell, and proteolytically processed for activation and also requiresglycosaminoglycans for activation of specific signaling pathways. TheGDNF amino acid sequence contains two potential glycosylation sites(discussed in greater detail below).

A multiple sequence alignment of the human GDNF isoforms is presentedbelow. Signal sequences are depicted in bold. Signal peptides are aa1-19, aa 1-19, and aa 1-36 for isoforms 1, 2 and 3, respectively. Maturepeptides are depicted in italics. Mature peptides are aa 78-211, aa52-185, and aa 95-228 for isoforms 1, 2 and 3, respectively. GDNFproteins have a key functional domain, termed the “transforming growthfactor beta (TGF-β) like domain. TGF-β-like domains are aa 118-211, aa92-185, and aa 135-228, for isoforms 1, 2 and 3, respectively.TGF-β-like domains are underlined.

CLUSTAL 2.1 multiple sequence alignment (SEQ ID NOs: 6-9) iso1-----------------MKLWDVVAVCLVLLHTASAFPLPAGKRPPEAPAEDRSLGRRRA 1so3MQSLPNSNGAAAGRDFKMKLWDVVAVCLVLLHTASAFPLPAGKRPPEAPAEDRSLGRRRA iso2-----------------MKLWDVVAVCLVLLHTASAFPLPAAN----------------- iso4MQSLPNSNGAAAGRDFKMKLWDVVAVCLVLLHTASAFPLPAAN-----------------                 ************************.: iso1PFALSSDSNMPEDYPDQFDDVMDFIQATIKRLKRSPDKQMAVLPRRERNRQAAAANPENS iso3PFALSSDSNMPEDYPDQFDDVMDFIQATIKRLKRSPDKQMAVLPRRERNRQAAAANPENS iso2---------MPEDYPDQFDDVMDFIQATIKRLKRSPDKQMAVLPRRERNRQAAAANPENS 1so4---------MPEDYPDQFDDVMDFIQATIKRLKRSPDKQMAVLPRRERNRQAAAANPENS         *************************************************** iso1RGKGRRGQRGKNRG CVLTAIHLNVTDLGLGYETKEELIFRYCSGSCDAAETTYDKILKNL iso3RGKGRRGQRGKNRG CVLTAIHLNVTDLGLGYETKEELIFRYCSGSCDAAETTYDKILKNL iso2RGKGRRGQRGKNRG CVLTAIHLNVTDLGLGYETKEELIFRYCSGSCDAAETTYDKILKNL iso4RGKGRRGQRGKNRG CVLTAIHLNVTDLGLGYETKEELIFRYCSGSCDAAETTYDKILKNL************************************************************ iso1SRNRRLVSDKVGQACCRPIAFDDDLSFLDDNLVYHILRKHSAKRCGCI iso3SRNRRLVSDKVGQACCRPIAFDDDLSFLDDNLVYHILRKHSAKRCGCI iso2SRNRRLVSDKVGQACCRPIAFDDDLSFLDDNLVYHILRKHSAKRCGCI iso4SRNRRLVSDKVGQACCRPIAFDDDLSFLDDNLVYHILRKHSAKRCGCI************************************************

As mentioned above, the GDNF gene encodes a highly conservedneurotrophic factor. GDNF orthologs share, for example, about 90-95%identity (or more), e.g., human and rat sharing 92% identity (10 aminoacids (aa) are different) and human and mouse sharing 94% identity (8amino acids are different) when comparing mature protein sequences;human and rat sharing 92% identity and human and mouse sharing 93%identity when comparing preproprotein sequences.

Pairwise alignments of human vs. mouse and human vs. rat preproproteinsare presented below. The glycosylation sites are Asn (=N) 126 and Asn162, and are indicated in bold. The skilled artisan will understand thata glycosylation motif is described as NX[ST] where X=any amino acid. Theglycosylation motifs in, for example, human GDNF (isoform 1) are asfollows aa126-128 (with N-linked glycosylation predicted to occur atN126, and at aa162-164 (with N-linked glycosylation predicted to occurat N162.) (See e.g., Lin et al., 1994, J. Neurochem, 63, 758-68; Truppet al., 1995, J. Cell Biol, 130, 137-48).

Hu   1 MKLWDVVAVCLVLLHTASAFPLPAGKRPPEAPAEDRSLGRRRAPFALSSDSNMPEDYPDQ  60MKLWDVVAVCLVLLHTASAFPLPAGKR  EAPAED SLG RR PFAL+SDSHMPEDYPDQ Mu   1MKLWDVVAVCLVLLHTASAFPLPAGKRLLEAPAEDHSLGHRRVPFALTSDSNMPEDYPDQ  60 Hu  61FDDVMDFIQATIKRLKRSPDKQMAVLPRRERNRQAAAANPENSRGKGRRGQRGKNRGCVL 120FDDVMDFIQATIKRLKRSPDKQ A LPRRERNRQAAAA+PENSRGKGRRGQRGKNRGCVL Mu  61FDDVMDFIOATIKRLKRSPDKQAAALPRRERNRQAAAASPENSRGKGRRGQRGKNRGCVL 120 Hu 121TAIHLNVTDLGLGYETKEELIFRYCSGSCDAAETTYDKILKNLSRNRRLVSDKVGQACCR 180TAIHLNVTDLGLGYETKEELIFRYCSGSC++AET YDKILKNLSR+RRL SDKVGQACCR Mu 121TAIHLNVTDLGLGYETKEELIFRYCSGSCESAETMYDKILKNLSRSRRLTSDKVGQACCR 180 Hu 181PIAFDDDLSFLDDNLVYHILRKHSAKRCGCI 211 P+AFDDDLSFLDDNLVYHILRNHSAKRCGCI Mu181 PVAFDDDLSFLDDNLVYHILRKHSAKRCGCI 211 Hu   1MKLWDVVAVCLVLLHTASAFPLPAGKRPPEAPAEDRSLGRRRAPFALSSDSNMPEDYPDQ  60MKLWDVVAVCLVLLHTASAFPLPAGKR  EAPAED SLG RR PFAL+SDSNMPEDYPDQ Ra   1MKLWDVVAVCLVLLHTASAFPLPAGKRLLEAPAEDHSLGHRRVPFALTSDSNMPEDYPDQ  60 Hu  61FDDVMDFIQATIKRLKRSPDKQMAVLPRRERNRQAAAANPENSRGKGRRGQRGKNRGCVL 120FDDVMDFIQATIKRLKRSPDKQ A LPRRERNRQAAAA+PENSRGKGRRGQRGKNRGCVL Ra  61FDDVMDFIQATIKRLKRSPDKQAAALPRRERNRQAAAASPENSRGKGRRGQRGKNRGCVL 120 Hu 121TAIHLNVTDLGLGYETKEELIFRYCSGSCDAAETTYDKILKNLSRNRRLVSDKVGQACCR 180TAIHLNVTDLGLGYETKEELIFRYCSGSC+AAET YDKILKNLSR+RRL SDKVGQACCR Ra 121TAIHLNVTDLGLGYETKEELIFRYCSGSCEAAETMYDKILKNLSRSRRLTSDKVGQACCR 180 Hu 181PIAFDDDLSFLDDNLVYHILRKHSAKRCGCI 211 P+AFDDDL FLDD+LVYHILRKHSAKRCGCI Ra181 PVAFDDDLWFLDDSLVYHILRKHSAKRCGCI 211

Human, mouse and rat GDN sequence are set forth as SEQ ID NOs: 6, 10 and11, respectively. The sequences appearing between aligned sequencesabove can be considered consensus sequences and are set forth as SEQ IDNOs:12-13 (where no match between amino acids in aligned sequences canbe depicted as X in a consensus sequence, X being one of the twomismatched residues, as depicted.

A multiple sequence alignment of human (isoform 1), mouse and rat GDNForthologs (mature proteins) is presented below:

Hu  78 SPDKQMAVLPRRERNRQAAAANPENSRGKGRRGQRGKNRGCVL 120 Mu  78SPDKQAAALPRRERNRQAAAASPENSRGKGRRGQRGKNRGCVL 120 Ra  78SPDKQAAALPRRERNRQAAAASPENSRGKGRRGQRGKNRGCVL 120 Hu 121TAIHLNVTDLGLGYETKEELIFRYCSGSCDAAETTYDKILKNLSRNRRLVSDKVGQACCR 180 Mu 121TAIHLNVTDLGLGYETKEELIFRYCSGSCESAETMYDKILKNLSRSRRLTSDKVGQACCR 180 Ra 121TAIHLNVTDLGLGYETKEELIFRYCSGSCEAAETMYDKILKNLSRSRRLTSDKVGQACCR 180 Hu 181PIAFDDDLSFLDDNLVYHILRKHSAKRCGCI 211 Mu 181PVAFDDDLSFLDDNLVYHILRKHSAKRCGCI 211 Ra 181PVAFDDDLWFLDDSLVYHILRKHSAKRCGCI 211

The above sequences are set forth as amino acids 78-211 of SEQ ID NOs:6, 10 and 11 respectively.

Mature protein sequences are also envisioned in which a methionine (Met;M) precedes the first amino acid of the mature sequence. The M can beadded for recombinant protein expression of mature proteins, and isencoded in engineered cDNA expression systems. However, the skilledartisan will appreciate that there are also expression systems whichallow the cleavage of the N-terminal M, as it can induce autoimmunereactions or changes in activity of the expressed, mature protein. Forexample see Nakagawal et al. 1987, Nature Biotech 5, 824-827;Fernández-San Millán et al., 2007, J Biotechnol. 20; 127(4):593-604;U.S. Pat. No. 4,870,017.

GDNF sequences are as described above and additional information on saidsequences can be found in the GenBank references indicated by thereferenced GenBank/gi reference numbers. GDNF sequences are alsodescribed in Science. 1993 May 21; 260(5111):1130-2; and in WO 93/06116and U.S. Pat. No. 7,226,758B1. Truncated forms are further described inUS20040127419(A1). Mutations of GDNF are described, for example inEketjäll et al. 1999, EMBO 8, 5901-5910. The GDNF protein is furtherdisclosed in, e.g., U.S. Pat. No. 6,362,319 and European Patent No. 0610 254, and a truncated form of GDNF in U.S. Pat. No. 6,184,200 andEuropean Patent No. 0 920 448.

Exemplary aspects of the invention can further include modified (e.g.,recombinantly-modified) forms of GDNF, or of biologically activefragments thereof. In one embodiment, the method of the inventionfeature the use of fusion proteins of GDNF, for example, fusion proteinsincluding serum albumin or a biologically active fragment thereof, e.g.,human serum albumin or a biologically active fragment thereof.) Furtherexemplary aspects of the invention feature pegylated GDNF proteins,glycan-modified GDNF proteins (e.g, having N-glycan integrated withinthe protein) and/or polymer-conjugated GDNF (e.g., polymers consistingof a polystyrene backbone with side chains of trehalose.)

Preferred aspects of the invention feature GDNF polypeptides, GDNFhomologs (e.g., GDNF orthologs) and/or biologically active portions(i.e., bioactive fragments) of GDNF polypeptides. In one embodiment,GDNF polypeptides can be isolated from cells or tissue sources by anappropriate purification scheme using standard protein purificationtechniques. GDNF polypeptide can be further derived from said isolatedpolypeptides using standard enzymatic techniques. In another embodiment,GDNF polypeptides or bioactive fragments thereof are produced byrecombinant DNA techniques. Alternative to recombinant expression, GDNFpolypeptides or bioactive fragments thereof can be synthesizedchemically using standard peptide synthesis techniques.

Polypeptides of the invention are preferably “isolated” or “purified”.The terms “isolated” and “purified” are used interchangeably herein.“Isolated” or “purified” means that the protein or polypeptide issubstantially free of cellular material or other contaminating proteinsfrom the cell or tissue source from which the polypeptide is derived,substantially free of other protein fragments, for example, non-desiredfragments in a digestion mixture, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations in whichthe polypeptide is separated from other components of the cells fromwhich it is isolated or recombinantly produced. In one embodiment, thelanguage “substantially free of cellular material” includes preparationsof polypeptide having less than about 30% (by dry weight) of non-GDNFpolypeptide (also referred to herein as a “contaminating protein”), morepreferably less than about 20% of non-GDNF polypeptide, still morepreferably less than about 10% of non-GDNF polypeptide, and mostpreferably less than about 5% non-GDNF polypeptide. When the polypeptideor protein is recombinantly produced, it is also preferablysubstantially free of culture medium, i.e., culture medium representsless than about 20%, more preferably less than about 10%, and mostpreferably less than about 5% of the volume of the polypeptidepreparation. When the polypeptide or protein is produced by, forexample, chemical or enzymatic processing from isolated or purified GDNFprotein, the preparation is preferably free of enzyme reactioncomponents or chemical reaction components and is free of non-desiredGDNF forms, e.g., aggregates, or GDNF fragments, i.e., the desiredpolypeptide represents at least 75% (by dry weight) of the preparation,preferably at least 80%, more preferably at least 85%, and even morepreferably at least 90%, 95%, 99% or more or the preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of polypeptide in which the polypeptideis separated from chemical precursors or other chemicals which areinvolved in the synthesis of the polypeptide. In one embodiment, thelanguage “substantially free of chemical precursors or other chemicals”includes preparations having less than about 30% (by dry weight) ofchemical precursors or reagents, more preferably less than about 20%chemical precursors or reagents, still more preferably less than about10% chemical precursors or reagents, and most preferably less than about5% chemical precursors or reagents.

Bioactive fragments of GDNF polypeptides include polypeptides comprisingamino acid sequences sufficiently identical to or derived from the aminoacid sequence of the GDNF protein, respectively, which include lessamino acids than the full length protein, and exhibit at least onebiological activity of the full-length protein. Typically, biologicallyactive portions comprise a domain or motif with at least one activity ofthe full-length protein. A biologically active portion of a GDNFpolypeptide can be a polypeptide which is, for example, 10-20, 20-30,30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-120, 120-140,140-160, 160-200, or more amino acids in length. In a preferredembodiment, a bioactive portion of a GDNF protein comprises a TGFβ-likedomain. Moreover, other biologically active portions, in which otherregions of the protein are deleted, can be prepared by recombinanttechniques and evaluated for one or more of the functional activities ofa native GDNF protein. Mutants of GDNF can also be utilized as assayreagents or therapeutic or pharmaceutical agents, for example, mutantshaving reduced, enhanced or otherwise altered biological propertiesidentified according to one of the activity assays described herein.

As defined herein, a GDNF polypeptide of the invention includespolypeptides having the amino acid sequences set forth above, as well ashomologs and/or orthologs of said polypeptides, i.e. polypeptides havingsufficient sequence identity to function in the same manner as thedescribed polypeptides. To determine the percent identity of two aminoacid sequences (or of two nucleotide or amino acid sequences), thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in the first sequence or second sequence for optimalalignment). The amino acid residues or nucleotides at correspondingamino acid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same residue as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % homology=# of identical positions/total # ofpositions×100), optionally penalizing the score for the number of gapsintroduced and/or length of gaps introduced.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In one embodiment, the alignment generated over a certainportion of the sequence aligned having sufficient identity but not overportions having low degree of identity (i.e., a local alignment). Apreferred, non-limiting example of a local alignment algorithm utilizedfor the comparison of sequences is the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithmis incorporated into the BLAST programs (version 2.0) of Altschul, etal. (1990) J. Mol. Biol. 215:403-10. BLAST alignments can be generatedand percent identity calculated using BLAST protein searches (e.g., theXBLAST program) using GDNF protein, or a portion thereof as a query,score=50, wordlength=3.

In another embodiment, the alignment is optimized by introducingappropriate gaps and percent identity is determined over the length ofthe aligned sequences (i.e., a gapped alignment). To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Research25(17):3389-3402. In another embodiment, the alignment is optimized byintroducing appropriate gaps and percent identity is determined over theentire length of the sequences aligned (i.e., a global alignment). Apreferred, non-limiting example of a mathematical algorithm utilized forthe global comparison of sequences is the algorithm of Myers and Miller,CABIOS (1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

A GDNF bioactive fragment is any fragment of GDNF having sufficient sizeand structure to carry out at least one activity (e.g., biologicalactivity) of the corresponding full-length GDNF protein. Exemplarybioactive fragments include, but are not limited to, enzymatic domains,protein binding and/or interaction domains, receptor binding domains,and the like. Preferred bioactive fragments include regions or domainscomprising a TGFβ-like domain, as defined herein.

The GDNF protein or GDNF bioactive fragment, may be detectably labeled.As defined herein, a protein or protein fragment which is “detectablylabeled” is on which has been modified to include a component detectableby standard laboratory means, e.g., the protein has been radioactivelylabeled, chromogenically labeled, or fluorescently labeled. Labeling canbe direct, i.e., protein is modified to directly contain the detectablelabel, or can be indirect, e.g., the protein is modified to include acomponent with which the detectable label interacts. Furthermore, inother embodiments, the activity of a GDNF protein or GDNF bioactivefragment may be compared to an appropriate control.

In preferred embodiments, a GDNF polypeptide or homolog or bioactivefragment thereof includes at least a TGFβ-like, as defined herein. TheTGFβ-like domain is a domain conserved among most, if not all TGFβfamily members. To identify the presence of a TGFβ-like domain in anGDNF polypeptide, the amino acid sequence of the polypeptide can besearched against a database of conserved protein domains (e.g., the CDdatabase at the NCBI) using the default parameters (Marchler-Bauer A etal. (2013), “CDD: conserved domains and protein three-dimensionalstructure.”, Nucleic Acids Res. 41(D1):D384-52.). NCBI Conserved DomainsDatabase Accession number pfam00019 sets forth a conserved TGFβ-likedomain amino acid sequence.

In exemplary embodiments, a TGFβ-like domain includes about 90-110 aminoacid residues, e.g., about 90-100 or 91, 92, 93, 94, 95, 96, 97, 98, 99or 100 amino acid residues). In exemplary embodiments, a domainidentification score of 60 is a suitable threshold score for determiningthe presence of the domain. For example, searches using the amino acidsequences of human GDNF (isoform 1), mouse GDNF and rat GDNF wereperformed against the CD database resulting in the identification of aTGFβ-like domain at amino acids 118-211 in each protein.

In preferred embodiments, a GDNF composition is a mature human GDNFprotein consisting of 134 amino acids, i.e., amino acids 78-211 of SEQID NO:6. This sequence contains two putative N-glycosylation sites aswell as seven conserved cysteines in the same relative spacing as theother members of the TGF-beta protein family (Lin et al. 1993, Science,260: 1130-1132; Eigenbrot and Gerber, 1997, Nat Struct Biol, 4:435-438;Chang et al. 2002, Endocri Rev, 23:787-823). Biologically active matureGDNF dimer is formed by a covalent disulfide bond between the unpairedcysteines in the monomers (Eigenbrot and Gerber, 1997, Nat Struct Biol,4:435-438).

In other exemplary embodiments, a GDNF protein for use in the methods ofthe invention can be a variant GDNF protein (or polypeptide), e.g., avariant having at least 90% or at least 95% or more identity. Preferredare biologically active variant GDNF polypeptides. As used herein, thephrase “biologically active variant GDNF polypeptide” refers to a GDNFpolypeptide that, when dimerized, binds to a ternary complex containingGFRα1 and RET. Any method for detecting binding to this complex can beused to evaluate the biological activity a variant GDNF polypeptide.Exemplary assays for detecting the ternary complex-binding ability of avariant GDNF polypeptide are described in WO00/01815. Variant GDNFpolypeptides can also be assayed or tested for their ability to triggera GDNF signaling cascade. For example, a kinase receptor activation(KIRA) assay can be used to assess the ability of a variant GDNFpolypeptide to induce RET autophosphorylation (see e.g., Sadick et al.,1996, Anal. Biochem., 235(2): 207) or assays can be performed to detectexpression of downstream targets of a GDNF signaling cascade, e.g.,increased expression of ret or fgfr2.

III. Wound Healing

The body's response to skin injury is focused on rapid wound closure,restraining invasion of microorganisms, and preventing excessive fluidloss (Singer A J, et al., N Engl J Med 341:738-46, 1999; Aarabi S, etal., PLoS Med 4:e234, 2007; Gurtner G C, et al., Nature 453:314-21,2008; Mustoe T., Am J Surg 187:65S-70S, 2004).

An increased understanding of the molecular mechanisms that regulate thevarious events of wound healing has laid the foundation for therapeuticinterventions attempting to improve the healing outcome. The cell-celland cell-matrix interactions are fundamental for successful woundhealing, and growth factors and cytokines maintain the balance ofsignals that regulate cellular migration, proliferation, and adhesion toa large extent. Freedberg I M, et al., J Invest Dermatol 116:633-40,2001; Hantash B M, et al., Front Biosci 13:51-61, 2008; Werner S, etal., Physiol Rev 83:835-70, 2003; Barrientos S, et al., Wound RepairRegen 16:585-601, 2008. Malfunction leads to a prolonged healing time orcomplete failure to heal and may result in a chronic wound. The woundfluid from chronic wounds has an increased concentration ofproinflammatory cytokines in comparison with wound fluid from acutewounds. Bennett N T, et al., Am J Surg 166:74-81, 1993; Robson M C, etal., Arch Surg 135:773-7, 2000. By contrast, there is a decreasedconcentration of growth factors in chronic wounds with high proteaseactivity and decreased levels of natural protease inhibitors. Nwomeh BC, et al., Clin Plast Surg 25:341-56, 1998; Mast B A, et al., WoundRepair Regen 4:411-20, 1996; Tarnuzzer R W, et al., Wound Repair Regen4:321-5, 1996. This deficiency in growth factors can be attributable todecreased production or secretion, more rapid breakdown, and, as is thecase in venous stasis ulcers, binding to in macromolecules, making themnonfunctional. Robson M C, et al., Arch Surg 135:773-7, 2000; Falange V,et al., Lancet 341:1006-8, 1993.

Glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN), andtheir receptors, GDNF family receptor α-1 (GFRα-1) and GDNF familyreceptor α-2 (GFRα-2), are critically important for development insystems such as kidney and nervous system. Moreover, Gdnf has been shownto be expressed in embryonic skin where Gdnf mRNA is detected in bothepithelial and mesenchymal components (Hellmich et al. 1996, Mech Dev54, 95-105). However, the role of these factors (e.g., GDNF) in skinbiology, and in particular, wound healing, is as yet not fullyunderstood. Knowledge of the role of this important factor in skinbiology would be helpful in understanding not only the various normalwound-healing events but also those occurring under distinctpathological conditions, for example, conditions in which wound healingis impaired, e.g., pathological conditions such as diabetes. Inaddition, development of effective novel therapies for wound healing canbased, at least in part, on this better understand of the effect of GDNFon the total wound-healing process. This approach facilitates thedevelopment of new products with potential applications in wound healingand other area of skin biology.

In humans, and more widely in all mammalian species, the wound-healingprocess can be subdivided into three consecutive and overlapping phases:inflammation, tissue formation, and matrix formation and remodeling.Rodero, M. P., et al., Int. J. Clin. Exp. Pathol. 3:643653, 2010. Thetransition from one phase to another depends on the maturation anddifferentiation of the main cell populations involved, among whichkeratinocytes, fibroblasts, neutrophils, and macrophages play the mainroles. Rodero, M. P., et al., Int. J. Clin. Exp. Pathol. 3:643653, 2010;Leibovich, S. J., et al., Am. J. Pathol. 78:71-100, 1975; Deonarine, K.,et al., J. Transl. Med. 5:11, 2011; Becker, D. L., et al., Biochim.Biophys Acta 1818:2068-2075, 2011. Recent observations show that stemcells have an unclear but likely major role in response to cutaneousinjury, Lanza R., Handbook of Stem Cells. Academic Press, 2004, as wellas the evidence for the roles of M1 and M2 macrophage, and that of Tcells. Gilliver, S. C., et al., Exp. Dermatol. 20:1-6, 2011; Sindrilaru,A., et al., J. Clin. Invest. 121:985-997, 2011.

Initial stages of wound healing involve the formation of a blood clotand inflammation. The inflammatory response is followed by proliferationand migration of dermal and epidermal cells, and matrix synthesis, inorder to fill the wound gap and reestablish the skin barrier (Cotran, etal., 1999; Hackam, D. J., et al. Surg Infect 3 (Suppl 1), S23-5 (2002);Harding, K. G., et al. Int Wound J 2, 364-8 (2005)). Finally, tissueremodeling and differentiation enable full recovery of the skin tissueand restoration of skin aesthetics (Hackam, D. J., et al. Surg Infect 3(Suppl 1), S23-5 (2002); Diegelmann, R. F., et al. Front Biosci 9, 283-9(2004)). The consensus in the literature is that the stepwise process ofwound healing first strives toward immediate filling of the gap,followed by re-epithelialization and reestablishment of the skin barrier(Yamaguchi, Y., et al. J Dermatol 28, 521-34, (2001)).

The early response is activated immediately after injury, resulting inthe inflammatory phase (first stage) of wound healing. Grose R, et al.,Mol Biotechnol 28:147-66, 2004. After hemostasis a fibrin clot isformed, which later serves as a scaffold for infiltrating cells. Inaddition, neutrophils and monocytes are recruited to the wound inresponse to trauma and bacterial contamination. Martin P, et al., TrendsCell Biol 15:599-607, 2005. In detail, the first event occurring afterinjury is the formation of a blood clot; several cells are involved inthe blood plug: platelets, and red and white blood cells. With theaction of fibrin fibers, the clot is stabilized and then “invaded” byseveral infiltrating cells, such as neutrophils, macrophages,mastocytes, platelets, and, possibly, by bacteria and toxins, which arecounteracted by host-generated H₂O₂. Neutrophils massively infiltratingthe wound during the first 24 hours postinjury are attracted by numerousinflammatory cytokines produced by activated platelets, endothelialcells, as well as by degradation products from pathogens. Macrophagesmassively infiltrating the wound two days postinjury produce intensephagocytic activity. Mosser, D. M., et al., Nature Rev. Immunol.8:958-969, 2008.

The second stage of wound repair (tissue formation) occurs approximately2 to 10 days after the injury and is characterized by proliferation andmigration of different cell types. Keratinocytes migrate over the woundbed while fibroblasts and macrophages replace the fibrin clot withgranulation tissue. Werner S, et al., J Invest Dermatol 27:998-1008,2007. The newly formed immature dermis is neovascularized, and thekeratinocytes behind the leading edge proliferate and differentiate torestore the barrier function of the epidermis. In detail, after two tothree days, the second phase lasts about two weeks and is characterizedby neo-angiogenesis and granulation. During the re-epithelializationprocess, keratinocytes from the wound edges migrate over the wound bedat the interface between the wound dermis and the fibrin clot. Thismigration is facilitated by the production of specific proteases, suchas collagenase by the epidermal cells to degrade the extracellularmatrix. Activated fibroblasts also migrate to the wound bed and form,with the macrophages, granulation tissue. Intense angiogenesis, allowingthe supply of oxygen and nutrients necessary for the healing process,also occurs within the tissue. Both growth factors and reactive oxygenspecies (ROS) produced by the granulation tissue will favorproliferation and differentiation of epithelial cells, restoringepithelial barrier integrity

Tissue remodeling, the third stage of wound repair, begins 2 to 3 weeksafter injury and lasts for 1 year or more. The type III collagen that isdeposited in the initial stages of wound healing is slowly replaced bytype I collagen, thereby forming the mature dermis. Loworn H N 3^(rd),et al., J Pediatr Surg 34:218-23, 1999. In detail, The last stage of thewound-healing process consists in a gradual involution of thegranulation tissue and dermal regeneration. This step is associated withapoptosis of myofibroblasts, endothelial cells, and macrophages. Theremaining tissue is therefore composed mostly of extracellular matrixproteins, essentially collagen type III that will be remodeled bymetalloproteinase produced by epidermal cells, endothelial cells,fibroblasts, and the macrophages remaining in the scar and then replacedby collagen type I. Singer, A. J., et al., N. Engl. J. Med. 341:738-746,1999.

IV. Hair Growth

As is the case for wound healing, the role of Glial cell line-derivedneurotrophic factor (GDNF) and its receptors in hair growth control, isas yet not fully understood. As mentioned above, Gdnf has been shown tobe expresses in embryonic skin where Gdnf mRNA is detected in bothepithelial and mesenchymal components (Hellmich et al. 1996, Mech Dev54, 95-105). Gdnf has also been shown to be expressed during human hairfollicle development (Adly et al. 2008 J Am Acad Dermatol, 58:238-250)and has further been shown to be expressed in adult mice in hairfollicles during the anagen to catagen transition phase (Botchkareva etal. 2000, Am J Pathol 156, 1041-1053). These gene expression patternssuggest a role for GDNF in the hair follicle development cycle. Theinstant inventors discovered that in a transgenic mouse model, when Gdnfis over-expressed under the Cathespin L promoter, it affects the hairfollicle growth in mice. Further analysis suggested that the effect ofGDNF on the hair follicles in these mice was possibly due to expressionof Gdnf in cells expressing endogenous Cathespin L gene, cells of theouter and inner root sheath and is essential for regular hair folliclemorphogenesis and cycling.

Hair grows in cycles of various phases: anagen is the growth phase;catagen is the involuting or regressing phase (also termed“apoptosis-driven regression”); and telogen, the resting or quiescentphase (see e.g., Stenn and Paus (2001) “Controls of Hair FollicleCycling”. Physiological Reviews 81 (1): 449-494 and Paus et al., “Thebiology of hair follicles”, NEJM 1999, 341:491-497). The time thesephases last varies from person to person. Different hair color andfollicle shape affects the timings of these phases. anagen phase, 2-3years (e.g., approximately 3 years, occasionally much longer); catagenphase, 2-3 weeks; and telogen phase, around 3 months.

Each phase has several morphologically and histologicallydistinguishable sub-phases. Prior to the start of cycling is a phase offollicular morphogenesis (formation of the follicle). There is also ashedding phase, or exogen, that is independent of anagen and telogen inwhich one of several hair that might arise from a single follicle exits.Normally up to 90% of the hair follicles are in anagen phase while,5-15% (or 10-14%) are in telogen and 1-2% in catagen. The cycle's lengthcan vary depending on location on different parts of the body.

Anagen is the active growth phase of hair follicles. The root of thehair are dividing rapidly, adding to the hair shaft. During this phasethe hair grows about 1 cm every 28 days. Scalp hair stays in this activephase of growth for 2-7 years. The amount of time the hair folliclestays in the anagen phase is genetically determined. At the end of theanagen phase an unknown signal causes the follicle to go into thecatagen phase.

The catagen phase is a short transition stage that occurs at the end ofthe anagen phase. It signals the end of the active growth of a hair.This phase lasts for about 2-3 weeks while the hair converts to a clubhair. A club hair is formed during the catagen phase when the part ofthe hair follicle in contact with the lower portion of the hair becomesattached to the hair shaft. This process cuts the hair off from itsblood supply and from the cells that produce new hair. When a club hairis completely formed, about a 2 week process, the hair follicle entersthe telogen phase.

The telogen phase is the resting phase of the hair follicle. Duringtelogen, the resting hair remains in the follicle until it is pushed outby growth of a new anagen hair. In most people, 5-15% of the hair on thescalp is in telogen at any given time. Shedding does not occur until thenew anagen hairs begin to grow. The emerging hairs help to force theresting hairs out of the follicle. Recent evidence suggests that themechanism of shedding of a telogen hair is an active process that mayoccur independent of the emerging anagen hair. When the body issubjected to extreme stress, as much as 70 percent of hair canprematurely enter a phase of rest, called the telogen phase. This hairbegins to fall, causing a noticeable loss of hair. This condition iscalled telogen effluvium (see below). Telogen effluvium is a form ofnonscarring alopecia characterized by diffuse hair shedding, often withan acute onset. Telogen effluvium can affect hair on all parts of thebody, but, generally, only loss of scalp hair is symptomatic.Understanding the pathophysiology of telogen effluvium requiresknowledge of the hair growth cycle (as detailed herein.) The club hairis the final product of a hair follicle in the telogen stage, and is adead, fully keratinized hair. Fifty to one-hundred club hair are sheddaily from a normal scalp.

The symptom of both acute and chronic telogen effluvium is increasedhair shedding and diffuse hair loss from the entire scalp. Acute telogeneffluvium is defined as hair shedding lasting less than 6 months.Patients usually only complain that their hair is falling out at anincreased rate or that the remaining hair feels less dense. Causes fortelogen effluvium and acute hair shedding can be physiologic stress,papulosquamous diseases of the scalp such as psoriasis and seborrheicdermatitis, allergic contact dermatitis, immunizations, severeinfections (HIV), acute illness such as febrile illness, major surgeryand severe trauma as well as chronic illness such as malignancy,particularly lymphoproliferative malignancy, systemic lupuserythematosus, end-stage renal disease, or liver disease, hormonalchanges such as pregnancy and delivery (can affect both mother andchild), hypothyroidism, discontinuation of estrogen-containingmedications; changes in diet like crash dieting, anorexia, low proteinintake, and chronic iron deficiency, heavy metals such as selenium,arsenic, and thallium. Acute telogen effluvium can occur in either sex,but because hormonal changes in the postpartum period are a common causeof telogen effluvium, women may have a greater tendency to experiencethis condition. Patients with acute telogen effluvium usually complainof relatively sudden onset of hair loss. If greater than 25% ofextracted hairs are in telogen, the diagnosis of telogen effluvium isconfirmed. However, each patient's scalp hair has an individualcharacteristic growth cycle. There are patients who have a very longanagen phase and a small proportion of hair in telogen at any giventime. Telogen effluvium can be caused by medications, such asbeta-blockers, anticoagulants, retinoids (including excess vitamin A),propylthiouracil (induces hypothyroidism), carbamazepine, andimmunizations.

V. Methods of Treatment

The instant invention features the use of GDNF for treatment in caseswhere wound healing and/or hair growth is desired. In one embodiment,the invention features a method of promoting cutaneous wound healing ina subject, which includes the step of administering at a wound site onthe subject a composition comprising a therapeutically effective dose ofGDNF, or a biologically active fragment thereof, and (optionally)repeating the administration for a time period sufficient to promotesaid cutaneous wound healing.

As defined herein, the phrase “cutaneous wound healing” refers to woundhealing of the skin, in particular, the skin of a mammal. “Cutaneouswound healing” is also referred to in the art as “dermal wound healing.”Cutaneous wound healing is quite distinct from, for example, cornealwound healing. For example, the phases and sequence of events occurringin these type of wound healings differ. Moreover, the goals of thesetypes of wound healing differ, for example, in the desired endpoint.Notably, one of the most crucial aspects of corneal wound healing is howthe healing process aims to minimize end results such as vascularizationand scar formation (which would have serious visual consequences). Bycontrast, such processes are significant desired end results of woundhealing in other parts of the body, in particular, vascularization.

Administration is preferably topical administration but can also beachieved by injection of GDNF compositions of the invention at the woundsite. In preferred embodiments, the wound site is external, e.g., on orin the skin of the subject. In exemplary embodiments, the wound site isan incision, a laceration, an abrasion, a puncture wound, a penetrationwound, a surgical wound, an ulceration, a burn, a contusion, a hematoma,or a crush injury. In the case of topical administration, a GDNFcomposition of the invention can further include at least oneanti-inflammatory agent. Alternatively, a GDNF composition of theinvention can further include at least one antibiotic. Alternatively, aGDNF composition of the invention can further include at least one otherwound healing-promoting agent.

In exemplary aspects of the invention, administration of GDNFcomposition is repeated for a time at least sufficient to promotefilling and re-epithelialization of a wound site. In preferred aspectsof the invention, administration of GDNF composition is repeated for atime at least sufficient to reestablish a skin barrier at the woundsite.

In exemplary aspects, formulations of GDNF can contain at least about 1ng/ml and up to about 10 μg/ml (e.g., for cosmetic applications), atleast about 10 μg/ml and up to about 100 μg mg/ml or even 1 mg/ml (e.g.,for wound healing and/or hair growth applications). In other exemplaryaspects, a therapeutically effective dose consists of an amount of GDNFadministered in a set period of time (e.g., daily), and routinelyrepeated over time (e.g., over weeks or months) to get the desiredtherapeutic effect. For example, GDNF compositions can be used at theconcentration recited above and administered in a therapeuticallyeffective dose (e.g., 1-100 ng daily, 100 ng to 1 μg daily, 1-50,50-100, or 100-500 μg daily, 100 or 500 μg, up to 1 mg daily, 1-10 mgdaily, 10-20 mg daily, 20-30 mg daily, 30-40 mg daily, or more. In someembodiments, a GDNF composition is administered daily. In otherembodiments, a GDNF composition is administered multiple times a daye.g., twice or three times daily. The skilled artisan will readilyappreciate that doses can be significantly lower if the compositions areto be administered via a controlled release system or formulation. Forexample, doses in the ng/ml or even pg/ml range are possible in the caseof controlled release systems or formulations.

In other aspects, the invention features methods for promoting hairgrowth on a subject which includes the step of administering at site ofdesired hair growth on the subject (e.g., at a skin site where hairfollicles grow) (e.g., on a scalp) a composition comprising apharmaceutically effective dose of isolated GDNF, or a biologicallyactive fragment thereof, and repeating the administration for a timeperiod sufficient to promote said hair growth on said subject.

As a measure of efficacy, a pharmaceutically effective dose is a dosesufficient to promote a 5%, 10%, 15%, 20%, 25% increase in hair folliclenumber (or follicular units) over a period of several weeks to severalmonths. Preferably, a pharmaceutically effective dose is a dosesufficient to promote a 10% increase in hair follicle number (orfollicular units) over a period of several weeks to several months. Evenmore preferably, a pharmaceutically effective dose is a dose sufficientto promote a 50% (or more) increase in hair follicle number (orfollicular units) over a period of several weeks to several months. Inexemplary embodiments, the method of promoting hair growth involvesadministration of a GDNF composition via topical contacting at a skinsite containing hair follicles (or a skin site that normally containshair follicles). A skin site for hair growth can be virtually anywhereon the body except, for example, the soles of the feet and the palms ofthe hands, the lips, and the eyelids, apart from eyelashes.

As an alternative, efficacy of treatment can be monitored according toany art-recognized means for evaluating hair loss in a subject. Withoutbeing bound in theory, it is proposed that the methods of the inventioncan reduce hair shedding, for example, to a normal level. Exemplarynon-invasive methods for monitoring hair loss include daily hair counts,standardized wash test, and the like (e.g., questionnaires, 60-s haircount, global photographs, dermoscopy, hair weight, contrasting feltexamination, hair feathering test, phototrichogram and TrichoScan),which are good methods for primary evaluation of the patient and to getan approximate assessment of the amount of shedding. While notpreferred, semi-invasive methods, e.g., Trichogram and unit areatrichogram (UAT), and/or invasive methods, e.g., scalp biopsy, can alsobe used to measure hair loss (and, indirectly, hair growth.). For adetailed description of these procedures, see e.g., Dhurat, R. andSaraogi, P. 2009 Int J Trichology 1(2): 108-119.

As a measure of efficacy, a pharmaceutically effective dose is a dosesufficient to promote a 5%, 10%, 15%, 20%, 25% decrease in hair lossover a period of several weeks to several months. Preferably, apharmaceutically effective dose is a dose sufficient to promote a 10%decrease in hair loss over a period of several weeks to several months.Even more preferably, a pharmaceutically effective dose is a dosesufficient to promote a 50% (or more) decrease in hair loss over aperiod of several weeks to several months.

In exemplary embodiments, the hair growth promoting methods of theinvention are used to treat androgenetic alopecia (AGA), also known asmale pattern baldness or female pattern baldness. In other embodiments,the hair growth promoting methods of the invention are used to treatautoimmune alopecia. This disease interferes with the hair growth cycleby causing a follicle to prematurely leave the anagen, or active growth,phase and enter the resting, or telogen phase. The hair growth in theaffected follicles is lessened or stopped completely.

In other embodiments, the hair growth promoting methods of the inventionare used to treat hair shedding. In other embodiments, the hair growthpromoting methods of the invention are used to treat hair thinning. Inyet other embodiments, the methods of the invention are used to treatacute or chronic telogen effluvium. In still other embodiments, themethods of the invention are used, generally, to treat scalp hair loss,hair thinning or baldness (poor hair thickness, poor hair growth). Inyet another embodiment, the methods of the invention are used to extendthe life or otherwise improve the condition of hair implants.

In other embodiments, the methods of the invention are used to treatiatrogenically-induced hair loss, for example, scalp hair and/or eyebrow hair loss in chemotherapy patients. As used herein, the phrase“iatrogenically-induced” refers to a condition or disease state causedby a physician, surgeon, or other administering professional or by amedical or surgical treatment (e.g., pharmaceutical treatment,chemotherapeutic treatment, radiation treatment) or a diagnosticprocedure.

In some embodiments, the GDNF compositions of the invention can be usedin combination with one or more additional agents, e.g., art-recognizedagents that promote wounds healing or hair growth and/or reduce hairloss. For example, the GDNF compositions of the invention might be usedin combination, or in a combination therapy, with any one (or more thanone) of the following agents: Combination platelet-derived growth factor(PDGF), interleukin-1 (IL1), nerve growth factor (NGF) or proNGF,keratinocyte growth factor (KGF), thymic peptides of the families ofthymulin, thymosin alpha-1 and thymosin beta-4 (see e.g.,US20110281802A1). Combination treatment can include, in otherembodiments, coincident oral treatment with, for example, vitamins;combination of vitamin B1, vitamin B6, vitamin B12, folic acid,magnesium glycinate, L-cysteine, biotin, ferric glycinate, Polygonummultiflorum, and/or Emblica officinalis (see e.g., US20130017285);insulin, insulin-like growth factor (IGF) (see e.g. US20100172865; Yoonet al. 2011, PLoS ONE 6(12): e28474), and/or polyamines (see e.g., Ramotet al. 2011. PLoS ONE 6(7): e22564). In some embodiments, the GDNFcompositions of the invention can be used in combination with anelectrical stimulus or mechanical stimulus (see e.g., Yoon et al. 2011,PLoS ONE.)

Without being bound in theory, it is also believed that GDNF, or abiologically active fragment thereof, can be administered to subjectsvia a gene therapy approach, for example, in cases where healing ofchronic wounds is desired. GDNF-encoding nucleic acids can be engineeredinto appropriate expression vectors and targeted to areas requiringcontinued supply of expressed GDNF. Expression vectors can be engineeredto encode GDNF, or a biologically active fragment thereof, in thecontext of a fusion protein, for example, fuse with a receptor targetingmeans for achieving entry into cells of the skin. Also, vectors can beformulated with agents that promote entry of nucleic acid molecules intoskin.

VI. Other Uses

Chronic Wound Healing

The instant invention also features the use of GDNF for treatment incases where a subject suffers from chronic nonhealing wounds. Expertsdebate about the time for closure that defines a chronic nonhealingwound as compared to that required for closure of an acute wound.Dealey, C., 3rd ed. Blackwell Publishing Ltd., 2005; Whitney, J. D.,Nurs. Clin. North Am. 40:191-205, 2005; Bryant, R A., et al., Acute andChronic Wounds, Current Management Concepts. 4th ed. Mosby, 2011. It hasbeen stated that “acute wounds generally follow trauma or inflammationand usually heal within six weeks.” Kumar, S., et al., Surgery 26:43-47,2008. Chronic wounds (in addition to failing to heal after six weeks)have characteristic pathological associations that inhibit or delay thehealing process. Jones. K. R., et al., Adv. Skin Wound Care 20:591-600,2007.

Chronic wound healing is mainly sustained by chronic inflammation, whichwithout appropriate therapy tends to worsen. The basic reasons are notnecessarily old age but rather hypertension and atherosclerosis, whichcan lead to ischemia, diabetes, and venous insufficiency. Commonpathogenetic causes are local tissue hypoxia, edema, abundant bacterialcolonization, and, possibly, repeated ischemia-reperfusion injuries. Thesurface area of a nonhealing wound tends to widen and shows fibrindeposition, necrotic areas, and a few islands of granulation tissue. Itis estimated that, in the industrialized world, 1-1.5% of the populationexperience problems related to recovering proper skin function. Theproblem is particularly prominent in elderly and diabetic patients, orthose with arteriosclerosis.

Common chronic wounds include, but are not limited to pressure ulcers,venous ulcers, and the like. Pressure ulcers, also known as decubitusulcers or bedsores, are localized injuries to the skin and/or underlyingtissue usually over a bony prominence, as a result of pressure, orpressure in combination with shear and/or friction. Most commonly thiswill be the sacrum, coccyx, heels or the hips, but other sites such asthe elbows, knees, ankles or the back of the cranium can be affected.The cause of pressure ulcers is pressure applied to soft tissue so thatblood flow to the soft tissue is completely or partially obstructed.Shear is also a cause; shear pulls on blood vessels that feed the skin.Pressure ulcers most commonly develop in persons who are not movingabout or are confined to wheelchairs. Pressure ulcers can be verydifficult to prevent in critically ill patients, frail elders,wheelchair users (especially where spinal injury is involved) andterminally ill patients. Venous ulcers (stasis ulcers, varicose ulcers,or ulcus cruris) are wounds that are thought to occur due to improperfunctioning of venous valves, usually of the legs. The exact etiology ofvenous ulcers is not certain, but they are thought to arise when venousvalves that exist to prevent backflow of blood do not function properly,causing the pressure in veins to increase. They are a major cause ofchronic wounds, occurring in 70% to 90% of chronic wound cases. Venousulcers develop mostly along the medial distal leg, and can be verypainful.

Burn Wounds

The instant invention also features the use of GDNF for treatment incases where a subject suffers from burn wounds. Most burn wounds affectonly the skin (epidermal tissue). Rarely, deeper tissues, such asmuscle, bone, and blood vessels can also be injured. Burns are describedaccording to the depth of injury to the dermis and are looselyclassified into first (involving the epidermis), second (extending intosuperficial (papillary) dermis and/or extending into deep (reticular)dermis), third (extending through entire dermis), and fourth (extendingthrough skin, subcutaneous tissue and into underlying muscle and bone)degrees. Burns are caused by a wide variety of substances and externalsources such as exposure to chemicals, friction, electricity, radiation,and heat. Generally, the methods of the invention are suited to thetreatment of first through third degree burns.

Freezing Injury

The instant invention also features the use of GDNF for treatment incases where a subject suffers from freezing injury, in particular, fromwounds or tissue damage resulting from freezing injury. An exemplaryfreezing injury is frostbite. Frostbite is the medical condition inwhich localized damage is caused to skin and other tissues due tofreezing. Frostbite is most common in body parts farthest from the heartand those with large exposed areas. Frostbite involves tissuedestruction. Second-degree injury usually involves blisters (appearing1-2 days after tissue becoming frozen.) The blisters may become hard andblackened, with time. Most of the injuries heal in one month, but thearea may become permanently insensitive to both heat and cold. The GDNFtreatment methodologies of the invention are suited for treatment oftissue damage, blisters, wounds and the like associated with frostbiteand other freezing injuries.

Diabetics

Diabetes mellitus is well known for its skin complications, usuallyleading to the formation of chronic debilitating ulcers (Levin, M. E.1995, DiabetesCare 18, 1383-94; Cavanagh, P. R., et al. 1998, OstomyWound Manage 44, 6S-12S; Brem et al., 2003). Wound-healing impairment ischaracterized by the inability of the healing process to progress, thusleaving the wound susceptible to external infections as well as todeterioration of the underlying tissue, leading to morbidity andsometimes requires amputation (Brem, H. et al. Surg Technol Int 11,161-7 (2003); Freedman, H., et al. Am J Surg 188, 31-5 (2004); Mousley,M. Nurs Times 99, 70-4 (2003); Wertheimer, E. Isr Med Assoc J 6, 287-9(2004)). Accordingly, the instant invention also features the use ofGDNF for treatment in cases where a subject suffers from ulcersassociated with diabetes. In exemplary embodiments, the inventionfeatures use of GDNF for treatment of “diabetic foot ulcer” and/or“non-healing chronic diabetic ulcers.”

Cosmetic Applications

Without being bound in theory, it is proposed that the GDNF compositionsof the invention may have utility in the field of cosmetic applications,e.g., in dermatological application. For example, the GDNF compositionsof the invention could be useful as. “antiaging” substances and/or mayimprove the appearance of wrinkles. It was noted in the wound healingexperiments, described herein, that mice exhibited smoother skinfollowing GDNF treatment. Other research on wound healing has producedmuch evidence showing the importance of peptides in improving the signsof aging (Lupo M P, Cole A L. Dermatol Ther. 2007:20:343-349). Thus, inexemplary embodiments, the GDNF peptides of the invention can be used inmethods to improve fine lines, skin texture, and/or hyperpigmentation inaddition to their uses to influence wound healing. It is important forsuch application that the/protein peptide is stable in formula,deliverable to its target dermal site, and biologically active at thistarget site (Lupo M. Dermatol Surg. 2005; 31:832-836).

Screening Assays

In other aspects, the invention features the use of GDNFpeptides/proteins in screening assays, e.g., in screening assays forcompounds that modulate one or more of the biological activities of GDNFin the processes of wound healing and/or hair growth. In one embodiment,the invention features contacting a composition comprising GDNF with atest compound and determining the ability of the test compound toupregulate, e.g., increase or enhance, the activity of GDNF (or abiologically active fragment thereof) such that a compound having thepotential to increase hair growth or improve wound healing isidentified. In another embodiment, the invention features contacting acell expressing GNDF with a test compound and determining the ability ofthe test compound to upregulate, e.g., increase or enhance, theexpression or activity of GDNF (or a biologically active fragmentthereof) such that a compound having the potential to increase hairgrowth or improve wound healing is identified. In exemplary embodiments,the cell is a cell known in the art to play a role in wound healingand/or hair growth. A multitude of screening assay formats art known inthe art and it is contemplated that screening assays of the inventioncan make use of labeled reagents, e.g., labeled GDNF proteins, unlabeledreagents, immobilized reagents, and the like. High throughput formatsare also well known in the art and are contemplated as a preferredembodiment for the screening assays of the invention.

VI. Pharmaceutical Compositions and Formulations

In other aspects, the invention features pharmaceutical formulationsthat include a therapeutically effective dose of isolated GDNF, or abiologically active fragment thereof, formulated in combination with atleast one agent which facilitates administration of said GDNF, or abiologically active fragment thereof.

Topical formulations are often prepared in the form of emulsions. Theterm “emulsion,” as used herein refers to mixtures of two or moreliquids, which may be in the form of a continuous phase and a dispersephase, for example. Exemplary emulsions may be in the form of creams,lotions, ointments, gels, etc. and may include, for example,oil-in-water emulsions, water-in-oil emulsions, multiple emulsions andmicroemulsions. These formulations will be prepared which contain fromabout 0.001 to 10 w/w % of the GDNF compositions of the presentinvention. These formulations will then be administered or applied tothe desired areas, e.g., from 1 to 4 times daily. Alternatively, theseformulations will be administered or applied to the desired areas lessfrequently, i.e., from 1 to 5 times a week. Formulations can be appliedor administered, for example, every other day, every third day, and soforth. Administration or application may vary in frequency over thecourse of treatment.

The GDNF compositions may also be administered topically in the form ofliposome delivery systems, such as small unilamellar vesicles, largeunilamellar vesicles and multilamellar vesicles. Liposomes can be formedfrom a variety of phospholipids, such as cholesterol, stearylamine orphosphatidylcholines. A potential formulation for topical delivery ofthe hair treatment compositions used in the methods of the presentinvention utilizes liposomes such as described in U.S. Pat. Nos.4,911,928 and 5,834,014.

Carriers for systemic administration include, for example, sugars,starches, cellulose and its derivatives, malt, gelatin, talc, calciumsulfate, vegetable oils, synthetic oils, polyols, alginic acid,phosphate buffer solutions, emulsifiers, isotonic saline andpyrogen-free water. Suitable carriers for parenteral administrationinclude, for example, propylene glycol, ethyl oleate, pyrrolidone,ethanol, and sesame oil.

In exemplary embodiments, the GDNF compositions of the invention areformulated in gels or in nanoparticles. Exemplary gels include, forexample, carboxymethylcellulose-based gels. Further exemplary gelformulations include, but are not limited to, polymeric gelformulations, in particular, those comprising a polymer selected fromthe group consisting of vinyl polymers, polyoxyethylene-polyoxypropylene copolymers, polysaccharides, proteins, poly(ethylene oxide),acrylamide polymers and derivatives or salts thereof. Such gelformulations are described in, e.g., U.S. Pat. No. 5,705,485;formulation with nanoparticles.

In other exemplary embodiments, the GDNF compositions of the inventionare formulated in heparin, heparan sulphate (see e.g., US Application2010/0056440), hyaluronic acid, lactic acid or in glycolic acid. Inother exemplary embodiments, the GDNF compositions of the invention areformulated in combination with polymeric compounds (such as polylacticacid, polyglycolic acid, poly(lactide-co-glycolide) (PLGA)microparticles, etc.) or in liposomes. In yet other exemplaryembodiments, the GDNF compositions of the invention are formulated incollagen-coated delivery systems or in combination with alginate,chitosan, lactide and/or lactide/glycolide copolymers. In yet otherembodiments, the GDNF compositions of the invention are formulated aspart of a topical dressing (e.g., within an adhesive bandage). In yetother embodiments, the GDNF compositions of the invention are formulatedas slow release forms, in films (e.g., biodegradable ornon-biodegradable firms. In yet other embodiments, the GDNF compositionsof the invention are formulated in combination with PEG 400 or serumalbumin (e.g., human serum albumin.)

In other exemplary embodiments, the GDNF compositions of the inventionare formulated as hydrogel compositions, i.e., hydrogels or hydrogelformulations. In a preferred aspect of the invention, the hydrogelscomprise GDNF. In a preferred aspect of the invention, the hydrogelscomprise GDNF included within liposomes. Liposomes have been used indelivering bioactive compounds, for example, growth factors and/orcytokines, for therapeutics purposes due to low toxicity, lack of immunesystem activation and targeted delivery at the site of action.

In one aspect, the invention features a hydrogel composed of liposomes(e.g., liposomes including GDNF) and chitosan. Ogiso et al., have shownthat the negatively charged liposomes diffuse to dermis and lower partof hair follicles, increasing the permeation of drug through the skin(Ogiso T, et al., (2001). J Drug Targeting.: 9, 49-59.) Chitosan, anatural polysaccharide polymer, has been used as a hydrogel mixed withliposomes to deliver growth factors at the injected site with slowrelease of the bioactive molecules. Earlier studies have shown thatproduction of the vascular endothelial growth factor is up-regulated inwound healing when macrophages are activated by chitin/chitosan (reviewby Muzzarelli, see e.g., Muzzarelli, R A A. (2009) CarbohydratePolymers: 76, 167-182). Moreover, Patel et al., have shown thatGDNF-Chitosan blended nerve guides enhances both functional and sensoryrecovery in vivo (Patel M, et al., (2007). J tissue Eng. & RegenerativeMed.: 1, 360-367). For further background, see e.g., Elcin Y M et al.(1996) Artif. Cell Blood Substit. Immobil. Biotechnol.: 24, 257-271

Accordingly, in one aspect, the invention features a therapeuticdelivery system, e.g., a drug delivery formulation, comprising liposomescontaining GDNF, formulated into a hydrogel, e.g., chitosan, foradministration in a therapeutic regimen described herein, for example,for administration to a wound site, e.g., in wound healing aspects ofthe invention. In an exemplary embodiment, the invention featureshydrogels made according to the following process. Growth factor orcytokine, e.g, GDNF is loaded into liposomes and then mixed withhydrogel agent, e.g., chitosan. Briefly, liposomes are dissolved inappropriate solvent or buffer and into a thin film (e.g., air and/or gasdried.) Dries films are then resuspended and filteres throughappropriately sized filters to generate small unilamellar vesicles.Liposome-encapsulated growth factor/cytokine, e.g, GDNF can be preparedby sonication of the lipids and growth factor/cytokine in appropriateweight/weight ration. To generate hydrogels, liposome-encapsulatedgrowth factor/cytokine, e.g, GDNF can be added to a solution, e.g., aprechilled solution) of hydrogel polymer, for example, chitosan(dissolved in appropriate solvent/buffer) by gentle stirring for anappropriate time before applying at the site of administration, e.g., atthe wound site.

It will be recognized by the skilled artisan that the compositionsso-formulated can also include inactive ingredients, for example,preservatives, stabilizers, solubilizers, and the like: sodium chloride,sodium acetate trihydrate, glacial acetic acid, water for injection, andmethylparaben, propylparaben, and m-cresol as preservatives and 1-lysinehydrochloride as a stabilizer.

Exemplary inactive ingredients include, for example, buffer, forexample, pH buffer e.g., sodium phosphate, potassium phosphate,histidine, or Tris-HCl, e.g., at a concentration of 10-50 mM; salt (forthe purpose of tonicity modifier and solubilizer), e.g., NaCl and CaCl₂at a concentration of 10-100 mM; sugar (for the purpose ofprotein-stabilizer. bulking agent, etc), e.g., sucrose or trehalose at aconcentration of 10-100 mg/mL; polyol (for the purpose of tonicitymodifier and bulking agent), e.g., mannitol or sorbitol at aconcentration of 10-100 mg/mL; amino acid (for the purpose of tonicitymodifier, bulking agent and stabilizer), e.g., glycine or arginine at aconcentration of 10-100 mg/mL; polymer (for the purpose of bulking agentetc.), e.g., hydroxyethyl starch at a concentration of 10-50 mg/mL;surfactant (for the purpose of solubilizer, stabilizer and aggregationinhibitor), e.g., Tween-80, Tween-40, and/or SDS at a concentration of<1 mg/mL; preservative (for the purpose of antimicrobial preservation),e.g., benzyl alcohol or phenol e.g., at a concentration of 1-10 mg/mL;and/or antioxidant (for the purpose of antioxidant), e.g., ascorbic acide.g., at a concentration of 1-10 mg/mL; and or other agents (with apurpose of protein-specific stabilization). Exemplary formulations arealso described, for example, in U.S. Pat. No. 8,383,114.

Administration may be by periodic injections of a bolus of thepharmaceutical composition or may be made more continuous by intravenousor intraperitoneal administration from a reservoir which is external(e.g., an IV bag) or internal (e.g., a bioerodible implant). See, e.g.,U.S. Pat. Nos. 4,407,957, 5,798,113, and 5,800,828, each incorporatedherein by reference.

Examples of parenteral delivery systems include, but are not limited to,ethylene-vinyl acetate copolymer particles, osmotic pumps, implantableinfusion systems, pump delivery, encapsulated cell delivery, liposomaldelivery, needle-delivered injection, needle-less injection, nebulizer,aeorosolizer, electroporation, hydrogels and transdermal patches. Insome embodiments, compositions of the invention may be provided inlyophilized form as a dried powder or a cake.

In the case of injections of the GDNF compositions, GDNF proteins can beformulated in sterile physiological saline solution (e.g., 10 nM citrateand 150 mM sodium chloride), optionally including heparin, heparansulphate, and/or glycerol.

In some embodiments, GDNF can be produced in Escherichia coli cells thatcontain an expression plasmid with a DNA insert encoding mature humanGDNF. In such embodiments, it is preferred to engineer into the proteinan N-terminal methionine.

The following examples illustrate the preparation of certain specificcompounds according to the present technology. A skilled artisanappreciates that the invention is not limited to the exemplary workdescribed or to the specific details set forth in the examples.

A skilled artisan further appreciates that the experimental conditionsdepicted in the following examples can be varied by as much as 2%, 5%,10% or 20% above or below the listed amount, temperature, concentration,pH, time and rpm in order to optimize the conditions to achieve thedesired results from the experiments.

EXAMPLES Example 1: Transgenic Overexpression of GDNF

In a transgenic mouse model generated in the laboratory for a differenthypothesis, when Gdnf is over-expressed under the Cathespin L promoter,it affects the hair follicle growth in mice. Further analysis suggestedthat the effect of GDNF on the hair follicles in these mice could be dueto expression of Gdnf in cells expressing endogenous Cathespin L gene,cells of the outer and inner root sheath and is essential for regularhair follicle morphogenesis and cycling. Though we do not understand themechanism how GDNF regulates hair follicle growth but Gdnf is expressedin embryonic skin where Gdnf mRNA is detected in both epithelial andmesenchymal components (Hellmich et al. 1996, Mech Dev 54: 95-105).

Transgenic mice were generated by microinjection a DNA constructcomprising the Cathespin L promoter operably linked to the Gdnf (DNACtsl promoter driven Gdnf transgene contains a 3 kb genomic fragmentupstream of the rat Ctsl translational start site. The numbering isrelative to the Ctsl transcriptional start site, designated by +1. Thecoding sequence of Gdnf-Gfp fusion gene was cloned downstream of thepromoter, using standard methods as described in Manipulating the MouseEmbryo: A Laboratory Manual, 3rd edition, Cold Spring Harbor LaboratoryPress; 2002, ISBN-10: 0879695919. The Cathespin L promoter is describedin Charron et al. 2003, Biol Reprod, 81(3), 1641-1648. When we analyzedthe FVB transgenic mice Tg(Ctsl-Gdnf) we detected ruffled fur by 3 wk ofage. We prepared paraffin skin sections stained with H&E. Analysis ofsuch revealed an increased number of hair follicles adjacent to normalhair development compared to skin section from control littermate. Thisphenotype was reproducible in a different genetic background, using themouse strain B6C3H. The transgenic mice expressing mouse Gdnf underCathespin L promoter (Tg-Ctsl-Gdnf) were generated in the FVBN/J andB6C3HF1 strain.

Example 2: Accelerated Wound Healing of a Full-Thickness Wound

Wild type C57BL/6J (B6) or BKS.Cg-Dock7^(m)+/+Lepr^(db) mice wereanesthetized using isoflurane. Before wound setting, the hair wasremoved on the dorsal side using a clipper. Loose hair was removed withdry gauze dampened with 70% ethanol. Four 3 mm full skin wounds weremade using a 3 mm biopsy punch needle. 100 μl of PBS or rat GDNFrecombinant protein (Ser78-Ile211) from R&D system (cat no. 512-GF) atconcentrations of 0.1 mg/ml or 0.5 mg/ml diluted in PBS was injectedeither at the wound site or subcutaneously in the middle of the 4 woundsusing 30 G needles, approximately 5-6 mm adjacent to the wounds. Themice were housed individually and monitored daily after surgery. Woundre-epithelialization and hair follicle development was determined byhistology at 96 hrs, 6, 8, and 15 days after injection of therecombinant GDNF protein (FIGS. 8A, 8B and 9).

Example 3: Dose Comparisons for Hair Follicles

For the GDNF injections we tested one dose of 100 μl 0.5 mg/ml GDNFprotein (R&D system, cat no. 512-GF) versus five 100 μl injections of0.1 mg/ml GDNF on alternate days.

Skin tissue was harvested 15 days after injection (day 15) and paraffinsections were prepared and stained with H&E. In both groups an increaseof hair follicles was detected, but a more striking increase when GDNFwas administered at the lower dose over five time points, with the hairfollicles being in different developmental stages (FIG. 3).

Example 4: Dose Comparison for Hair Follicle Development

Adult C57BL/6 (B6) wild type mice were injected with 100 μl of 0.1 mg/mlor 0.5 mg/ml recombinant GDNF protein (R&D system, cat no. 512-GF). Theskin was analyzed after 9 days. One injection of 0.1 mg/ml wassufficient to induce hair follicle development at the injection site byday 9 (FIG. 5A). With the higher dose of 0.5 mg/ml more hair follicleswere detectable (FIG. 5B). The rate of proliferation was assayed by5-Bromo-2′-deoxyuridine (BrdU) labeling (Sigma, cat. no. B5002)according the protocols described by Sanjay et al. 2008, Methods in MolBiol 438, 335-343. For this BrdU was injected intraperitoneally (i.p.)one day before collecting the skin Most of the new hair follicles in theGDNF group stained positively indicating that these are proliferatingwhile (data not shown). Alternatively skin sectioned were immunostainedwith Ki67 antibody, as Ki67 protein is an established marker for cellproliferation. Ki67 is expressed during active phase of the cell cycleG1, S and G2 and absent from resting cells G0. The staining of hairfollicle cells with Ki67 antibody shows that they are in proliferativestage on day 9 after GDNF injection (FIG. 5C).

Adult B6 mice were injected with 100 μl of PBS or 0.5 mg/ml of GDNF (allinjections were on the dorsal side and subcutaneous (s.c.)) and skin wasisolated at day 6, 9, 13, 15, 20, and 30 after injections, paraffinembedded and sectioned for histological analysis. A dramatic increase inhair follicle numbers was observed on day 9 and 13 compared to miceinjected with PBS (FIG. 6). By day 20 hair regeneration is complete inmice injected with PBS. Surprisingly, hair follicles are stillproliferating in the GDNF injected group (FIG. 6).

Example 5: GDNF Signaling in the Epidermis

We analyzed the effects of GDNF (R&D system, cat no. 512-GF) on skin byquantitative RT-PCR (q-RT-PCR). For this total RNA was extracted fromabout 100 mg, about 8 mm diameter, skin tissue isolated from areainjected either with PBS or 0.5 mg/ml GDNF protein 6 days afterinjection. RNA isolation was performed using Trizol Plus kit fromInvitrogen. qRT-PCR of specific genes listed below was performed usingOne step Real-time RT-PCR SYBR green kit from Applied biosystems. Betaactin was used as internal control. Transcripts of selected genes wereanalyzed and normalized to beta-actin. The PCR protocol used herefollowed the RT-PCR protocol according to Applied Biosystems One stepReal-time RT-PCR protocols. We tested expression of col4al, Ret, Fgfr2,c-myc, Notch1, Csf1 and Csfr1. A four-fold increase in Ret transcriptwas seen, and a three-fold increase for Notch1 and Csf1. For Ret andNotch1 one a role in hair growth has been described previously (Kato etal. 2001; Vauclair et al. 2005), and it may be that the action of GDNFis mediated by upregulating these. To date, it has not been shown thatNotch1 is part of the GDNF signaling pathway, and this is the first timeseeing this novel action of GDNF. We do not observe a change in c-myc,col4a1 and csfr1 expression. For example c-myc is not described as partof the GDNF signaling pathway and can be considered as negative control.While, it is known that c-myc overexpression results in proliferationepidermal cells, it does not seem to play a role here (FIG. 7).

The primers used for the q-RT-PCR are:

Fgfr2-Forward (SEQ ID NO: 14) 5′-ctctctacgtcatagttgaatatg-3Fgfr2-Reverse (SEQ ID NO: 15) 5′-atatccctggccaggccaaagtct-3′ Ret-Forward(SEQ ID NO: 16) 5′-agatgtttatgaggaagattccta-3 Ret-Reverse(SEQ ID NO: 17) 5′-Tcctcgctgcagagtctggcctc-3′ Col4a1-Forward(SEQ ID NO: 18) 5′-atgccctttctcttctgcaa-3′ Col4a1-Reverse(SEQ ID NO: 27) 5′-ctgcggaatctgaatggtct-3′ Csf1-Forward (SEQ ID NO: 19)5′-gatccctgagtctgtcttccacct-3′ Csf1-Reverse (SEQ ID NO: 20)5′-cagttccacctgtctgtcctcatcc-3′ Csf1r-Forward (SEQ ID NO: 21)5′-gtaaagtggatggccccagagagc-3 Csf1r-Reverse (SEQ ID NO: 22)5′-taggctccaggtcccagcaggactg-3′ c-Myc-Forward (SEQ ID NO: 23)5′-cagctcgcccaaatcctgtacctcgt-3′ c-Myc-Reverse (SEQ ID NO: 24)5′-cagacaccacatcaatttcttcctc-3′ Notch1-Forward (SEQ ID NO: 25)5′-tgaagaacggagccaacaaggacatgc-3′ Notch1-Reverse (SEQ ID NO: 26)5′-gcaatcggtccatgtgatccgtgatgt-3′

Example 6: GDNF Accelerates Wound Healing in B6 Mice

Mice were anesthetized using isoflurane and fur removed using clipperfrom the dorsal side. Loose fur was removed with dry gauze dampened with70% ethanol. Equal size 3 mm of full thickness wounds were set using abiopsy punch needle on the dorsal side of adult B6 mice. The mice werehoused individually and monitored daily after surgery. Wounds werephotographed on day 0 before injection and after one week, day 0 iscounted as day of injection. Only one wound site was injected once with100 μl of 0.5 mg/ml GDNF (R&D system, cat no. 512-GF) (arrow), the otherwound was injected with PBS (vehicle control). The wound site that wasinjected with 100 μl of 0.5 mg/ml GDNF healed faster than the woundsites injected with PBS. After one week mice were sacrificed and tissuearound the original wound was isolated to prepare sections. The sectionsof wound sites injected with PBS or GDNF were stained using H&E. By oneweek all layers of skin, epidermis, dermis and hair follicles arepresent at the wound site injected with GDNF compared to PBS injectedsites (FIG. 8A). In the PBS control the skin does not show the threelayers, but is still in the reconstruction phase after one week. Thewound repair starts as early as 48 hrs after the wound is set withimmune cells detectable. At the wound site red blood cell (RBC)infiltration is seen in both groups. but to a higher extent in the GDNFgroup (data not shown). By 96 hrs a complete layer of epithelium wasobserved throughout the wound site only for the GDNF group (FIG. 8Barrow) and many new blood vessels (arrowheads) are detectable in theGDNF injected site. In the PBS group less immune cells and blood vesselsare detectable compared to the GDNF group. At the one week time point nonew hair follicles are detectable in the PBS group.

Example 7: GDNF Accelerates Wound-Healing in Diabetic Mice

As diabetic model, we chose the diabetic mouse strainBKS.Cg-Dock7^(m)+/+Lepr^(db)/J (db/db; available from The JacksonLaboratory stock number 000642). Mice were anesthetized using isofluraneand fur removed using clipper from the dorsal side. Loose fur wasremoved with dry gauze dampened with 70% ethanol before 3 mm fullthickness wounds were set into the dorsal skin creating 4/wounds permouse using a biopsy punch needle. The mice were housed individually andmonitored daily after surgery. We treated the wounds either with 100 μlPBS or 100 μl of 0.5 mg/ml GDNF recombinant protein (R&D system, cat no.512-GF). Mice were sacrificed at several time points and tissues wascollected, fixed in formalin, sectioned and sections were stained withH&E. 96 hours after wound setting and GDNF treatment, we detectedneovascularization and blood vessel formation (FIG. 9A arrowhead) morepronounced in the GDNF group compared to the PBS group. The increase innew blood vessel formation in db/db mice treated with GDNF can be seenat the one week time point. Compared to wild type B6 mice wound healingin diabetes, takes two weeks instead of one week when injected with 0.5mg/ml of GDNF but at a higher dose (250 μl of 0.5 mg/ml GDNF or 2.5 foldmore). The healing process is greatly accelerated after GDNF treatmenteven in the db/db mice.

Example 8: GDNF Induces Blood Vessel Formation

FVB/NJ mice (n=5) (available from The Jackson Laboratory, cat no.001800) were s.c. injected with 100 μl of PBS or 100 μl of 0.5 mg/mlGDNF protein (R&D system, cat no. 512-GF). 9 days after the injectionthe mice were sacrificed and the skin was isolated, photographed andfixed in formalin for histology. When the skin was isolated it was verystriking that that the skin was showed more blood vessels with morebranching in the GDNF group (Fig. xx). Analyzing the H&E stainedsections confirmed an increased number of blood vessels around the GDNFinjection site in comparison to the PBS injected tissue.

Example 9: Hydrogel Formulations for Therapeutic Administration

An optimized formulation of liposome-encapsulated GDNF in hydrogel isprepared for the growth factor to be applied at the wound site. Thegrowth factor is loaded into the liposomes and mix with chitosan.Briefly, liposomes (from Sigma # L4395) are dissolved in 10 ml ofchloroform: methanol mixture (2:1) in round bottom flask and air driedto thin film by jet stream of argon gas at 40° C. The thin film is thenhydrated in water and passed through 0.2 um polycarbonate filters a fewtimes to get small unilamellar vesicles. The liposome-encapsulated GDNFis prepared by sonication of the lipids and growth factor in 0.250:1ratio (w/w) of growth factor to the lipid. These liposomes are added toa prechilled solution of chitosan (1.8% wt/vol in 2% acetic acid) bygentle stirring for 10 minutes before applying at the wound site.

The present invention and its embodiments have been described in detail.However, the scope of the present invention is not intended to belimited to the particular embodiments of any process, manufacture,composition of matter, compounds, means, methods, and/or steps describedin the specification. Various modifications, substitutions, andvariations can be made to the disclosed material without departing fromthe spirit and/or essential characteristics of the present invention.Accordingly, one of ordinary skill in the art will readily appreciatefrom the disclosure that later modifications, substitutions, and/orvariations performing substantially the same function or achievingsubstantially the same result as embodiments described herein can beutilized according to such related embodiments of the present invention.Thus, the following claims are intended to encompass within their scopemodifications, substitutions, and variations to processes, manufactures,compositions of matter, compounds, means, methods, and/or stepsdisclosed herein.

The contents of any patents, patent applications, and references citedthroughout the specification are herein incorporated by reference intheir entireties.

1. A pharmaceutical formulation comprising a dose of isolated glial cellderived growth factor (GDNF) polypeptide, or a biologically activefragment thereof, effective to promote cutaneous wound healing in asubject, in combination with at least one agent which facilitatesadministration of the polypeptide, or a biologically active fragmentthereof.
 2. The pharmaceutical formulation of claim 1, wherein thepolypeptide comprises the amino acid sequence of amino acids 78-211 ofSEQ ID NO:7 or the amino acid sequence of amino acids 118-211 of SEQ IDNO:7.
 3. The pharmaceutical formulation of claim 1, wherein thepolypeptide is encoded by the nucleic acid sequence comprisingnucleotides 201-836 of SEQ ID NO:1 or is encoded by a nucleic acidsequence having at least 90% or 95% identity to the nucleic acidsequence comprising nucleotides 201-836 of SEQ ID NO:1.
 4. Thepharmaceutical formulation of claim 1, wherein the polypeptide, orbiologically active fragment thereof is glycosylated at theglycosylation site occurring at amino acid residues 126-128 of SEQ IDNO:7 or at the glycosylation site occurring at amino acid residues162-164 of SEQ ID NO:7.
 5. The pharmaceutical formulation of claim 1,wherein the wound site is selected from the group consisting of anincision, a laceration, an abrasion, a puncture wound, a penetrationwound, a surgical wound, an ulceration, a burn, a contusion, a hematoma,and crush injury.
 6. The pharmaceutical formulation of claim 1, whereinthe formulation further comprises at least one anti-inflammatory agentor at least one antibiotic.
 7. The pharmaceutical formulation of claim1, wherein the formulation is administered to the subject at a dose ofat least about 0.1-0.5 mg/ml GDNF.
 8. The pharmaceutical formulation ofclaim 1, wherein the formulation is administered to the subject andadministration is repeated for a time sufficient to promote filling andre-epithelialization of a wound site; or administration is repeated fora time sufficient to reestablish a skin barrier at the wound site. 9.The pharmaceutical formulation of claim 1, wherein the formulation isadministered to the subject and administration is repeated for a timeperiod sufficient to promote cutaneous wound healing.
 10. Thepharmaceutical formulation of claim 9, wherein administration isrepeated daily, for at least 3-5 days; or repeated twice daily, for atleast 3-5 days.
 11. The pharmaceutical formulation of claim 1, whereinthe formulation is administered to the subject via injection at thewound site or via topical contacting at the wound site.
 12. Thepharmaceutical formulation of claim 1, wherein the subject has diabetes.13. A pharmaceutical formulation comprising a dose of isolated glialcell derived growth factor (GDNF) polypeptide, or a biologically activefragment thereof, effective to promote hair growth on a subject, incombination with at least one agent which facilitates administration ofthe polypeptide, or a biologically active fragment thereof.
 14. Thepharmaceutical formulation of claim 13, wherein the polypeptidecomprises the amino acid sequence of amino acids 78-211 of SEQ ID NO:7or the amino acid sequence of amino acids 118-211 of SEQ ID NO:7. 15.The pharmaceutical formulation of claim 13, wherein the dose is a dosesufficient to promote a 25% increase in hair follicle number or a 25%decrease in hair loss over a period of 3-4 months.
 16. Thepharmaceutical formulation of claim 13, wherein the formulation isadministered to the subject via topical contacting at the site ofdesired hair growth.
 17. The pharmaceutical formulation of claim 13,wherein the GDNF is glycosylated at the glycosylation site occurring atamino acid residues 126-128 of SEQ ID NO:7 or at the glycosylation siteoccurring at amino acid residues 162-164 of SEQ ID NO:7.
 18. Thepharmaceutical formulation of claim 13, wherein the dose consists of nomore than about 10 mg/ml GDNF.
 19. The pharmaceutical formulation ofclaim 13, wherein the formulation is administered to the subject and theadministration is repeated for a time period sufficient to promote hairgrowth.
 20. The pharmaceutical formulation of claim 19, wherein therepeating step is performed five times.